RU2469091C2 - Protein showing interferon-like antiviral and antiproliferative biological activities (versions), protein structure, coding polynucleotide (versions), expression vector, host cell, composition and use of protein as antiviral, antiproliferative, anticancer or immunomodulatory agent and method of treating condition sensitive to interferon-therapy, cancer or viral disease - Google Patents

Abstract

FIELD: medicine.

SUBSTANCE: what is produced is a recombinant protein created by gene shuffling possessing higher antiviral and antiproliferative activities as compared with existing human interferon-alpha-2b (HulFN-α2b). The prepared polypeptide is used in a composition as an antiviral, antiproliferative, anticancer or immunomodulatory agent.

EFFECT: invention enables using the prepared high-active interferon-like protein for treating a condition sensitive to interferon therapy, cancer or viral disease.

49 cl, 9 dwg, 8 tbl, 7 ex

Description

Field of Invention

This application relates to recombinant proteins having biological activities like human interferons.

State of the art

This application accepts the Interferon Nomenclature (IFN) published in Nature (1).

Human interferons (HuIFN), which were discovered by Isaacs and Lindenmann in 1957 (2), are a well-known family of cytokines secreted by a wide variety of eukaryotic cells under the influence of various stimuli, such as viral infection or mitogens. IFN can cause many changes in cellular behavior, including effects on cell growth and differentiation and modulation of the immune system (3-7). HuIFNs were classified into six subgroups, namely IFN-α, IFN-β, IFN-γ, IFN-ω, IFN-ε and IFN-κ. HuIFN-α (leukocyte interferon) is produced in human leukocytes and, together with minor amounts of HuIFN-β (interferon fibroblasts), in lymphoblastoid cells. In addition, according to their chemical and biological characteristics, HuIFNs were classified into two general categories, namely Type I and Type II. Type I consists of the subgroups IFN-α and INF-β, as well as the recently opened subgroups IFN-ω, IFN-ε and IFN-κ. Type II has only one member: IFN-γ (immune interferon).

Different subgroups of interferons have different structural and biological characteristics. HuIFN-β is an N-glycoprotein (8, 9), which has been purified to a homogeneous state and characterized. It is heterogeneous in size, mainly due to its carbohydrate moiety. However, there is only one human IFN-β gene encoding a protein of 166 amino acids. IFN-β has a low homology with IFN-α, exhibiting approximately 30-40% identity.

In contrast to the uniqueness of the IFN-β gene, HuIFN-α is a subgroup consisting of a multigenic family of essentially 14 genes. Minor variants formed by differences in one or two amino acids cause multiple alleles (10). Excluding the pseudogen 1FNAP22, we have 13 genes encoding 13 proteins. Each protein contains 165-166 amino acids. The protein encoded by the IFNA13 gene is identical to the IFNA1 protein. Thus, there are 12 individual interferon alpha proteins: IFNA1, IFNA2, IFNA4, IFNA5, IFNA6, IFNA7, IFNA8, IFNA10, IFNA14, IFNA16, IFNA17 and IFNA21. The amino acid sequence homology among IFN-α subtypes is usually 80-85% (11).

Mature IFN-ω demonstrates 60% homology of the nucleotide sequence with the family of IFN-α variants, but it is 6 amino acids longer at its C-terminus. IFN-ω is more distantly related to interferon-β (has approximately 30% sequence homology). Human IFN-ω is not classified in the IFN-α group, since it differs from IFN-α in its antigenic properties and differs in its interaction with IFN-α receptor, type I (12). IFN-ω is secreted by leukocyte-infected viruses as the main component of human leukocyte interferons.

The mature human IFN-ε protein contains 185 amino acids, having approximately 33% and 37% sequence homology with IFN-α2 and IFN-β, respectively (13, 14). The function and biophysical properties of IFN-ε are not substantially characterized in detail; however, it acts like interferons, type I. In addition, IFN-ε can play a role in the reproductive function (15).

The 180-amino acid IFN-κ human cytokine is a newly identified IFN, type I. The coding sequence of IFN-κ is approximately 30% identical to other interferons, type I, found in humans. A characteristic feature of IFN-κ is a noticeable constitutive expression of its transcript in uninduced cells, in particular keratinocytes. IFN-κ can play a role in the regulation of systemic or local immune functions through its effect on cells of the innate immune system (16). However, IFN-κ exhibits low antiviral activity (17).

Human interferon, type I, appears to bind to two receptor subunits, IFNAR-1 and -2, which are widely distributed on the cell surface of various cell types. The involvement of the ligand leads to the induction of phosphorylation of tyrosine kinases TYK2 and JAK-1 (Janus kinase 1), which are associated with IFNAR-1 and -2, respectively. After phosphorylation, STAT (signal transducer and activator of transcription) proteins are released from the complex with the receptor and form homodimers as well as heterodimers (18, 19). Upon release, STATA dimers associate with an interferon-sensitive factor 9 (IRF-9) that binds to a DNA protein to form a complex called IFN-stimulated gene factor-3 (ISGF-3), which migrates to the nucleus. Further, the ISGF-3 complex binds to an upstream DNA element in all IFN-inducible genes. This is the so-called “classic” signal transduction pathway.

Continuously discover new modes of action and biochemical pathways regulated by IFN, type I. For example, located downstream from P13K (phosphoinositide-3OH kinase) in the signal transduction pathway, nuclear factor Kappa-B (NF-κB) and PKC-d associated with anti-apoptotic effects observed in neutrophils incubated with IFN-β (20).

More than 300 genes, called interferon-induced genes, are sensitive to IFN. The most studied IFN proteins are those with antiviral properties. For example, an enzyme in the 2,5-oligosynthetase family (OAS-1 and -2) catalyzes the synthesis of short oligoadenylates, which bind to and activate RNase L (RNAseL), an enzyme that breaks down viral and cellular RNAs, thereby inhibiting protein synthesis. DC (double-stranded) -RNA-activated protein kinase (PKR) phosphorylates the translation initiation factor elF2a, which also leads to inhibition of the synthesis of viral and cellular proteins. More recently, PKR has also been found to be necessary for activation of the transcription factor NF-κB, a central active subject in the induction of inflammatory cytokines, immune modulation and apoptosis. Proteins MX (myxovirus resistance) inhibit viral RNA replication by either preventing the transport of viral particles in the cell, or transcribing viral RNA. RNA-specific adenosine deaminase (ADAR) converts adenosine into inosine, thus causing hypermutation of viral RNA genomes (21).

HuIFNs have a wide range of biological activities, including antiviral, antitumor, and immunoregulatory functions. The clinical capabilities of human interferons are extensively investigated and summarized below.

As regards antitumor applications, HuIFNs can mediate antitumor effects either indirectly by regulating immunomodulatory and anti-angiogenesis responses, or by directly affecting the proliferation or cell differentiation of tumor cells (22). Interferon therapy has been used in the treatment of various leukemia (23), for example, leukemic reticuloendotheliosis (24), acute and chronic myeloid leukemia (25-27), osteosarcoma (28), basal cell carcinoma (29), glioma (30), renal cell carcinoma (31) , multiple myeloma (32), melanoma (33), Kaposi’s sarcoma (23), and Hodgkin’s disease (34). Combination therapy using IFN-α with cytarabine (ara-C), 5-FU (5-fluorouracil), hydroxyurea and IL-2 (interleukin-2) has been well studied and in most cases shows significantly better results compared to therapy alone one HuIFN-α (3). A synergistic treatment of advanced cancer with a combination of HuIFN and temozolomide has also been reported (35).

As regards antiviral applications, HuIFN has been used clinically for antiviral therapy, for example, in the treatment of AIDS (36), viral hepatitis, including chronic hepatitis B, hepatitis C (37, 38), papilloma virus infection (39), herpes virus infections (40 ), viral encephalitis (41) and in the prevention of rhinitis and respiratory infections (40).

In addition, HuIFN has been used clinically for antibiotic therapy (42), for example, aerosolized HuIFN-γ (43) and HuIFN-α have been used in patients with multidrug-resistant pulmonary tuberculosis (44). HuIFN-γ has been used in the treatment of multidrug-resistant brain tuberculosis (45).

In addition, HuIFN has been used clinically for immunomodulatory therapy, for example, to prevent graft versus host rejection or to reduce the progression of autoimmune diseases such as multiple sclerosis (46, 47) and Sjogren's syndrome (48). IFN-β is approved by the FDA in the United States for the treatment of multiple sclerosis. Recently, there has been a report that in patients with multiple sclerosis the production of interferons type I and interleukin-2 is reduced (49). In addition, immuno-modulating therapy using HuIFN-α is likely to be an effective therapy in patients with chronic myeloid leukemia (CML) undergoing relapse after bone marrow transplantation (50).

As regards their use in vaccine formulations, HuIFNs have been used clinically as an adjuvant in the treatment of melanoma (51) and can also be used as an adjuvant or adjuvant to enhance or model the immune response in cases of prophylactic or therapeutic vaccination for many other diseases (52) .

HuIFN-α2a was the first angiogenesis inhibitor used in clinical trials and has been shown to be effective in children for the treatment of life-threatening hemangiomas (53, 54). Another clinical indication is a giant cell tumor of the bone. Kaban et al reported a striking regression of a large, rapidly growing, recurring giant cell tumor of the lower jaw (55).

Although HuIFNs have many important clinical uses, they exhibit significant side effects and other limitations. Most cytokines, including HuIFN, have relatively short half-lives from the bloodstream, as they are induced in vivo to produce local and short-term effects. Since usually HuIFN is administered as systemic therapeutic agents, they need to be administered frequently and in relatively large doses. Frequent parenteral administration is uncomfortable and painful. In addition, the toxic side effects associated with the administration of HuIFN are often so severe that some people cannot tolerate such treatment. In all likelihood, these side effects are associated with systemic administration of high dosages. In addition, in clinical studies it was found that some patients produce antibodies against rHuIFN (“r” from recombinant), which neutralizes its biological activity (56).

There is no doubt that the development of new interferon proteins with increased efficacy is an urgent need for numerous applications, for example, anticancer therapies, as well as antiviral, immunotherapy, antiparasitic, antibacterial therapy, or for any medical condition or situation where increased interferon activity and / or reduced side effects are required. effects. In general, it is highly likely that HuIFN will play a major role in the next generation of new antitumor and antiviral therapies (10).

It is well known in the art that most effective methods for improving the pharmaceutical properties of cytokine drugs are to modify the cytokine protein itself. During this time, various strategies and techniques for changing interferon peptides have been established. Typically, three strategies are currently used to create HuIFN-α mutants.

The first strategy is to get IFN hybrids. Some researchers took advantage of the presence of naturally occurring restriction endonuclease cleavage (RE) cleavage sites in IFN coding sequences to combine homologous coding fragments (57, 58). A review of a series of hybrid IFNs is provided by Horisberger and Di Marco (11); This article gives a general idea of how to construct such molecules. Specific examples of methods for constructing hybrid interferons are described. Some researchers have taken advantage of PCR amplification to construct mutant IFN-α in order to create specifically desired nucleic acid fragments and then improve the capabilities of new pieces of different IFNs joined together (59). US Pat. No. 6,685,933 (60) also describes PCR amplification techniques for creating human IFN hybrids. Interferon hybrids can be created within the interferon subgroup, as, for example, described in US patent No. 5137720 (61) and US patent No. 6685933 (60), or between at least two different interferon classification groups, as, for example, described in US patent No. 6,174,996 (62) and US Pat. No. 6,685,933 (60). In addition, the original hybrid genes can come from one species (mainly from humans), for example hybrids between HuIFN-α and HuIFN-ω, or from more than one animal species, for example hybrids between human and mouse interferon-α (63 )

A second strategy for constructing interferon mutants is to use site-directed point mutagenesis by introducing changes in one or more nucleotides into IFN DNA molecules (64). Recently, directed mutation and computational methods have been used as a guiding principle of protein mutagenesis (65).

The third strategy for constructing HuIFN, type I, is to shuffle (shuffling; from the English. Shuffling) fragments of the IFN gene obtained by digesting their RE, PCR amplification, chemical synthesis or DNAse digestion, followed by PCR for randomly connecting the fragments together and then their amplification. The resulting PCR products are in fact a pool of rearranged fragments of the interferon-alpha gene that can be used to construct a DNA library from which DNA clones with the desired phenotypes can be isolated (66). For example, Chang et al. Described a method for constructing and screening a shuffling library for HuIFN to identify HuIFN derivatives with increased antiviral and antiproliferative activity in mouse cells (67).

Human interferon-alpha-con-1 (consensus interferon) is a naturally-recombinant non-existent HuIFN-α of 166 amino acids. It was created by evaluating the most highly conserved amino acids in each respective region based on the known cloned HuIFN-α sequences. It has 89% sequence homology at the amino acid level with respect to HuIFN-α2b and a specific antiviral activity of approximately 10 ⁹ IU (international units) / mg. Human interferon-alpha-con-1 has been tested to treat chronic HCV infection (hepatitis C virus) in patients 18 years of age or older with compensated liver disease (68).

Despite the fact that some recombinant interferon proteins are known in the prior art, there is a need for new interferon-like proteins and protein compositions having enhanced biological activities.

SUMMARY OF THE INVENTION

According to the invention, an isolated polynucleotide encoding a protein having biological activities like human interferons is described. In one embodiment, the polynucleotide comprises a nucleotide sequence at least 93% identical to SEQ ID No: 1. In other embodiments, the nucleotide sequence is at least 95% identical to or at least 98% identical to SEQ ID No: 1.

In one embodiment, the invention includes a protein selected from the group consisting of proteins, each of which has an amino acid sequence of at least 85% identical to SEQ ID No: 2. Preferably, the protein is a non-naturally occurring protein and has increased antiviral and antiproliferative activity compared to human interferon-alpha-2b (HuIFN-α2b). For example, a protein can have an antiviral activity of at least 2 times that of HuIFN-α2b, and antiproliferative activity of at least 10 times that of HuIFN-α2b. In specific embodiments, the amino acid sequence of a given protein is at least 90% identical or at least 95% identical to SEQ ID No: 2

The present invention encompasses recombinant vectors containing the sequence of a given polynucleotide, and host cells containing these vectors. The invention also encompasses polypeptide fragments exhibiting biological activities like human interferons. In addition, the invention includes protein constructs and other compositions exhibiting interferon-like biological activities, such as conjugates containing a protein and another component, such as an inorganic polymer. In addition, the invention includes methods and uses of protein and compositions for therapeutic purposes, for example, as antiviral or anticancer agents. In addition, the invention can be used to treat other conditions that are sensitive to interferon therapy.

A brief description of the graphic materials

Figure 1 shows the complete DNA sequence encoding a new protein of the invention, referred to herein as Novaferon ™ (SEQ ID No: 1) (A). Figure 1 also shows the predicted amino acid sequence of Novaferon (SEQ ID No: 2) (B) and the alignment of this amino acid sequence of Novaferon with the DNA sequence of Novaferon (C). The first amino acid, cysteine, in the mature protein Novaferon is designated as residue 1.

Figure 2 shows the alignment of the nucleotide sequence of the Novaferon gene relative to the HuIFN-α14 gene (Genebank number: NM_002172) (A) and the amino acid sequence alignment of the Novaferon protein relative to the HuIFN-α14 protein (translated, Genebank number: NM_002172) (B). The first amino acid, cysteine, in the mature protein Novaferon is designated as residue 1. Novaferon has approximately 93% sequence identity (462/498) with HuIFN-α14 at the nucleotide level and approximately 87% sequence identity (144/166) at the amino acid level. Different nucleotides are shown as empty space in the middle of the line.

FIG. 3 shows the alignment of the nucleotide sequence of the Novaferon gene relative to the HuIFN-α2b gene (Genebank number: NM_000605) (A) and the amino acid sequence alignment of the Novaferon protein relative to the HuIFN-α2b protein (translated from the HuIFN-α2b gene with Genebank number: NM_000605) (AT). The first amino acid, cysteine, is designated as residue 1 in the mature Novaferon protein. Novaferon has approximately 89% sequence identity (445/498) with HuIFN-α2b at the nucleotide level and approximately 81% sequence identity (135/166) at the amino acid level. Different nucleotides are shown as empty space in the middle of the line.

Figure 4 is a graph showing antiproliferative inhibition of in vitro Daudi cells by Novaferon compared to HuIFN-α2b.

Fig. 5 is a graph showing the in vivo antitumor effects of Novaferon and HuIFN-α2b in athymic mice with human prostate cancer PC-3 xenografts.

6 is a graph showing the in vivo antitumor effects of Novaferon and HuIFN-α2b in athymic mice with human liver cancer Hep G2 xenografts.

FIG. 7 is a graph showing the in vivo antitumor effects of Novaferon and HuIFN-α2b in athymic mice with human melanoma A-375 xenografts.

FIG. 8 is a graph showing the in vivo antitumor effects of Novaferon and HuIFN-α2b in athymic mice with LS 180 colon cancer xenografts.

Fig. 9 is a graph showing the in vivo antitumor effects of Novaferon and HuIFN-α2b in athymic mice with human leukemia HL 60 (3) xenografts.

Detailed description

Throughout the following description, specific details are set forth in order to provide a more comprehensive understanding of the invention. However, the invention may be practiced without these details. In other cases, in order to avoid introducing unnecessary ambiguity into the invention, well-known factors are not shown or described in detail. Accordingly, the description and figures should be considered in an illustrative and not in a limiting sense.

Definition of Terms

Before giving a detailed description of the present invention, it should be understood that this invention is not limited to the specific protein molecules, methodology, protocols, cell lines, vectors and reagents described in all their possible diversity. It should also be understood that the terminology used in this description is intended only to describe specific embodiments and is not intended to limit the scope of the present invention, which will be limited only by the attached claims.

For a more complete understanding of the invention set forth in this description, the following terms are applied, and their definition is shown below. It should be understood that the terminology used in this description is intended only to describe specific embodiments and is not intended to limit the scope of the present invention, which will be limited only by the attached claims.

Unless otherwise specified, all technical and scientific terms used in this description have the same meanings that are commonly understood by one of ordinary skill in the art to which this invention corresponds. All publications mentioned in this description are incorporated into this description by reference to describe and disclose cell lines, vectors and methodologies, the reviews of which are given in these publications and which could be used in connection with this invention. Nothing in this description should be construed as an assumption that the invention gives the right to date such discoveries earlier on the basis of the previous invention.

The term "interferon" refers to a family of secreted proteins produced by a number of eukaryotic cells under the influence of various environmental stimuli, including viral infection or exposure to mitogen. In addition to possessing antiviral properties, interferons have been shown to affect a wide variety of cellular functions. All interferon units are expressed in this description with reference to international standards BO3 (World Health Organization) 94/786 (rHuIFN-α-consensus) and 95/650 (rHuIFN-α2a).

The term “interferon-like” (or “similar to interferon”) refers to functional and / or structural features demonstrated by known interferons or analogs of interferons or similar to those of known interferons or analogs of interferons. For example, the term “interferon-like biological activity” includes antiviral and antiproliferative activity. Other examples of interferon-like biological activities are provided herein, and will be apparent to those skilled in the art. The term “activity” in the plural includes the term “activity” in the singular; that is, the invention encompasses recombinant proteins or other protein constructs or compositions exhibiting at least one interferon-like activity.

The term "consensus interferon" refers to a type of synthetic interferon having an amino acid sequence representing an approximate averaging of the sequences of all known subtypes of human alpha interferons. It was reported that consensus interferon has a higher (approximately 5-fold) antiviral, antiproliferative and NK cell activation activity than any natural subtype of human IFN-α.

The term "isolated", as used herein, refers to molecules such as DNA or RNA that are extracted from their native environment. For example, recombinant DNA molecules contained in a vector are considered isolated for the purposes of the present invention. Additional examples of isolated DNA molecules include recombinant DNA molecules contained in heterologous host cells, or purified (partially or substantially) DNA molecules in solution. “Isolated” DNA also includes DNA molecules obtained from a library that may contain natural or artificial DNA fragments of interest, as well as chemically synthesized nucleic acids. Therefore, isolated nucleic acids can be obtained by recombinant technology.

The term “nucleotide sequence” refers to a nucleotide sequence containing an oligonucleotide, polynucleotide or nucleic acid molecule and fragments or parts thereof. In the case of a DNA molecule, the sequence may contain a series of deoxyribonucleotides, and in the case of an RNA molecule, the sequence may contain a corresponding series of ribonucleotides. An oligonucleotide, polynucleotide or nucleic acid molecule may be single or double stranded, and the nucleotide sequence may be a sense or antisense strand.

The terms “oligonucleotide fragment” or “polynucleotide fragment”, “part”, or “segment”, or “probe” or “primer” are used interchangeably and refer to a sequence of nucleotide residues with a length of at least about 5 nucleotides. Preferably, the fragments can be used for hybridization with the target nucleotide sequence. The primer serves as a nucleotide polymerization initiation site catalyzed by either DNA polymerase or RNA polymerase or reverse transcriptase. A fragment or segment can uniquely identify each polynucleotide sequence of the present invention. Preferably, the fragment contains a sequence substantially similar to SEQ ID No: 1.

The terms “protein” or “peptide” or “oligopeptide” or “polypeptide” refer to naturally occurring or synthetic molecules containing an amino acid sequence.

The term "open reading frame" or OPC means a series of nucleotide triplets for encoding amino acids without any termination codon and usually refers to a sequence translated into a protein.

The term "mature protein coding sequence" refers to a sequence that encodes a protein or peptide without a signal or leader sequence. This protein can be obtained by processing in the cell, which removes any leader / signal sequence. A protein can be obtained by synthesis or only by using a polynucleotide encoding a sequence encoding only a mature protein.

The terms “purified” or “substantially purified” as used herein mean that the protein is presented essentially in the absence of other biological macromolecules, for example, other proteins, polypeptides and the like. The protein is purified so that it is at least 95% by weight of the indicated biological macromolecules present (but water, buffers, and other small molecules, especially molecules with a molecular weight of less than 1000 daltons, may be present).

The term "recombinant expression carrier or vector" refers to a plasmid or phage or virus or vector for expression of a protein from a DNA sequence (RNA). The expression vector may contain a transcriptional unit containing a set of (1) genetic elements or elements that play a regulatory role in gene expression, e.g., promoters or enhancers, (2) a structural or coding sequence that is transcribed into mRNA and translated into a protein, and (3 ) the corresponding sequences of initiation and termination of transcription. Structural units intended for use in expression systems of yeast or eukaryotes preferably contain a leader sequence allowing extracellular secretion of the translated protein by the host cell. Alternatively, when a recombinant protein is expressed without a leader or transport sequence, it may contain a methionine residue at the amino terminus. This residue may subsequently or may not be cleaved from the expressed recombinant protein to give the final product.

The term "substantial similarity" refers to a nucleic acid or its fragment, which (s) has a high degree of sequence identity with another nucleic acid after its optimal alignment with another nucleic acid or its complementary strand. The sequence identity or homology can be determined using sequence analysis software, for example BLASTN. It is believed that the first nucleic acid has significant similarity with the second nucleic acid if they show sequence identity of at least about 85-95% or more after their optimal alignment. For example, to determine the identity or homology of a sequence between two different nucleic acids, the BLASTN program "BLAST (Basic Local Aligment Search Tool) 2 Sequences" is used. This program is available for public use from the National Center for Biotechnology Information (NCBI) (69). As a non-limiting example, such comparisons can be made using software installed in the default settings mode (expect = 10, filter = default, open gap = 5, extension gap = 2 penalties, gap x dropoff = 50). Similarly, the first protein or polypeptide is considered to have significant similarity with the second protein or polypeptide if they show sequence identity of at least about 85-95% or more after they are optimally aligned and compared using the BLAST (blastp) software with default settings .

As another illustration, a polynucleotide having a nucleotide sequence at least 95% "identical" to a reference nucleotide sequence encoding a protein means that the nucleotide sequence of a given polynucleotide is identical to the reference sequence except that the polynucleotide sequence may include up to five point mutations inclusive for every 100 nucleotides of the reference nucleotide sequence encoding a protein. In other words, to obtain a polynucleotide having a nucleotide sequence at least 95% identical to the reference nucleotide sequence, up to 5% of the nucleotides inclusive in the reference sequence can be delegated or replaced by another nucleotide or a series of nucleotides up to 5% inclusive of all nucleotides in the reference sequence can be inserted into a reference sequence.

The terms “complementary” or “complementarity”, as used herein, refer to the natural binding of polynucleotides by base pairing under the recommended salt and temperature conditions. For example, the sequence “A-G-T” binds to the complementary sequence “T-C-A”. The complementarity between two single-stranded molecules can be “partial”, with only a few nucleic acids being bound, or it can be complete when there is total complementarity between single-stranded molecules. The degree of complementarity between nucleic acid chains has significant effects on the efficiency and stringency of hybridization between nucleic acid chains. This is especially important in amplification reactions, which depend on the binding between nucleic acid chains.

The term "transformation" means the introduction of DNA into the body so that the DNA is capable of replication, either as an extrachromosomal element, or by chromosome integration. The term "transfection" refers to the delivery of an expression vector to a suitable host cell, regardless of whether or not any coding sequences are actually expressed.

The terms "treatment", "treatment" and their grammatical equivalents are used in a broad sense and include therapeutic treatment, prevention, prevention and alleviation of some undesirable symptoms or conditions.

The terms “biological activity” and “biological activities” as used herein refer to structural, regulatory, biochemical or other biological functions in living systems, for example, similar or identical to those of molecules existing or non-existent in nature.

The term "antiproliferation" and "antiproliferative", as used herein, refers to slowing down and / or preventing cell growth and division, resulting in a decrease in the total number of cells and / or a decrease in the percentage of target cells in any one or all phases of the cell cycle. In addition, cells can be specified as being in a state of arrest at a specific stage of the cell cycle: G1 (gap (gift) 1), S-phase (DNA synthesis), G2 (gap 2) or M-phase (mitosis). The term "antiproliferative activity", as used herein, refers to the activity of a protein, protein construct or composition that inhibits cell proliferation, especially neoplastic cell proliferation, such as cancer cells, either in vitro or in vivo.

The term “antitumor” or “anticancer,” as used herein, refers to counteracting or preventing the formation of malignant tumors. "Antitumor activity" or "anticancer activity," as used herein, refers to the activity of a protein, protein construct, or composition that inhibits cell proliferation, especially neoplastic cell proliferation, such as cancer cells, either in vitro or in vivo.

The term "IC ₅₀ " or "half maximum inhibitory concentration" is the concentration of an inhibitor, such as a protein, which is necessary for 50% inhibition of cell growth in vitro.

The terms “antiviral” and “anti-virus”, as used in this description, refer to the inhibition and / or prevention of viral infection of cells or the impact on the replication of the virus in cells in vitro and / or in vivo, leading to a slowdown or stop of the reproduction of the virus or decrease total number of viral particles. "Antiviral activity", as used herein, means the activity of a protein, protein construct or composition that inhibits viral infections or affects replication of the virus either in vitro and / or in vivo.

Protein Novaferon

The present invention relates to the preparation and characterization of a new human interferon-like protein, referred to herein as “Novaferon ™ ™. As described in detail below, Novaferon protein exhibits increased antiviral and antiproliferative biological activities compared to naturally occurring HuIFN-α2b, as measured by standard in vitro tests. In particular, Novaferon protein shows a 12.5-fold increase in antiviral activity when tested in the Wish-VSV system (Wish cells / vesicular stomatitis virus) and approximately 400-fold improvement in antiproliferative inhibition of Daudi cell growth compared to HuIFN-α2b in the same systems under test.

In one embodiment, the Novaferon protein is encoded by a polynucleotide consisting of 498 nucleotides, which are shown in SEQ ID No: 1 and Figure 1 (A). Mature Novaferon protein consists of 166 amino acids, which are shown in SEQ ID No: 2 and Figure 1 (B). Polynucleotide and amino acid sequences and their variants encompassed by the invention are described in further detail below.

For comparative purposes, the inventors investigated the homology of Novaferon with naturally occurring HuIFN. Using blast searches, Novaferon was found to have the highest homology with HuIFN-α14 at both the nucleotide and amino acid levels. As shown in FIG. 2, the polynucleotide sequence (SEQ ID No: 1) encoding Novaferon has approximately 93% homology (462/498) with respect to HuIFN-α14, and the amino acid sequence has approximately 87% homology (144/166) relative to HuIFN-α14. Compared to HuIFN-α2b, the most widely used human interferon product, homology is approximately 89% at the nucleotide level (445/498) and approximately 81% (135/166) at the amino acid level, as shown in FIG. 3.

Regarding synthetic IFN-alpha-con-1 (consensus interferon), Novaferon has approximately 91% sequence identity at the nucleotide level (453/498) and approximately 84% sequence identity at the amino acid level (140/166).

As described in detail in the experimental section below, the polynucleotide sequence (SEQ ID No: 1) was selected from the human interferon type I shuffling DNA library. Briefly, Novaferon protein was obtained by transfecting host cells with a recombinant vector containing the complete polynucleotide sequence of SEQ ID No: : 1. The Novaferon protein contained in the supernatant of the host cell line was purified, and it was shown that it exhibits biological activities similar to human interferons, such as antiviral and antiprol erativnuyu function.

Polynucleotide and Options

The new polynucleotide sequence / nucleic acid molecule of the present invention consists of 498 nucleotides, which are shown in Figure 1 (SEQ ID No: 1). Using the information provided herein, such as a nucleotide sequence, a nucleic acid molecule of the present invention encoding a Novaferon protein (SEQ ID No: 2) can be obtained by recombinant expression, chemical synthesis, or other standard molecular biology techniques such as as techniques for DNA mutagenesis.

The present invention, in addition to the isolated nucleic acid molecule (SEQ ID No: 1), also includes DNA molecules having sequences different from the DNA sequence described in SEQ ID No: 1, but, due to the degeneracy of the genetic code, still encoding that the same or essentially the same amino acid sequence of Novaferon protein (SEQ ID No: 2). Genetic codes and species-specific preferences for codons are well known in the art. Thus, it is common practice for a person skilled in the art to create degenerate variants of DNA sequences different from the DNA sequence from SEQ ID No: 1, for example, to optimize the expression of codons for a particular host (for example, to change the codons in human mRNA to those which are preferred for a bacterial host such as E. coli).

In addition, the invention provides an isolated nucleic acid molecule having the nucleotide sequence shown in FIG. 1 (SEQ ID No: 1), or a nucleic acid molecule having a sequence complementary to the nucleic acid sequence in SEQ ID No: 1. The present invention also provides the information on the following applies to recombinant vectors, including isolated nucleic acid molecules of the present invention, and to host cells containing recombinant vectors, and as well as methods for the manufacture of such vectors and the creation of host cells expressing Novaferon protein, and to the use of host cells to produce Novaferon by recombinant techniques.

Based on the nucleic acid sequence of the present invention (SEQ ID No: 1, Figure 1 (A)), this invention encompasses nucleic acid molecules that are substantially the same, such as nucleic acids having at least about 85-95% or more sequence identity with respect to SEQ ID No: 1 after their optimal alignment. For example, in one aspect, nucleic acids having approximately 93%, 95%, 96%, 97%, 98%, or 99% sequence identity with respect to the nucleotide sequence shown in SEQ ID No: 1 are within the scope of the invention, regardless whether they encode proteins or polypeptides having biological activities similar to Novaferon (such activities include, but are not limited to, increased antiviral, antiproliferative and antitumor functions compared to HuIFN). Such nucleic acid molecules could be used, for example, as probes for determining mRNA in cells already transfected with a vector containing the nucleotide sequence of the present invention to obtain Novaferon. In other words, these nucleic acid sequences, at least approximately 93%, 95%, 96%, 97%, 98% or 99% identical to the sequence shown in SEQ ID No: 1, could be used as markers for determining expression heterologous genes in the host cell.

In addition, the invention includes a polynucleotide containing any part of at least about 30 consecutive nucleotides, preferably of at least about 50 consecutive nucleotides, from SEQ ID No: 1.

More generally, this invention includes and encompasses fragments of any and all isolated nucleic acid molecules that are identical to the incomplete nucleic acid sequence (s) shown in FIG. 1 (SEQ ID No: 1). In one embodiment, the length of such fragments can be at least about 15 nucleotides, and they are useful as diagnostic probes and primers, which are discussed in this description. Moreover, this invention includes and encompasses longer fragments of approximately 50 nucleotides or more in length.

In addition to the nucleic acid sequence described in SEQ ID No: 1 encoding the Novaferon protein, the present invention also includes, but is not limited to, nucleic acid sequences encoding the amino acid sequence of the complete Novaferon protein together with additional amino acids / peptide (s) / polypeptide (s) ), for example, an added secretory leader sequence.

In addition, nucleic acid sequences having the nucleic acid sequence described in SEQ ID No: 1, as well as additional non-coding sequences, including, for example, but not limited to, introns and non-coding 5 ′ and 3 ′ sequences, are included in the invention. such as transcribed, untranslated sequences that play a role in transcription, mRNA processing (i.e. splicing and polyadenylation signals, binding to ribosomes and stabilization of mRNA), and additional coding sequences that encode additional amino acids with or without functionality.

In addition, the present invention relates to variants of the nucleic acid molecules of the present invention (SEQ ID No: 1), which encode parts, analogs or derivatives of Novaferon protein. Variants can be obtained by screening a shuffling library for interferons or using mutagenesis techniques and / or other known techniques described in the art.

As explained above, such variants may include variants obtained by insertion, deletion or substitution of nucleotides. Insertions, deletions or substitutions may affect one or more nucleotides. These mutations can occur at the 5'- or 3'-terminal positions of the reference nucleotide sequence or elsewhere between these terminal positions, scattered either individually among the nucleotides in the reference sequence, or in one or more consecutive groups within reference sequence. Changes can result in conservative or non-conservative amino acid substitutions, deletions, or additives. Especially preferred among them are silent substitutions, additives and / or deletions that do not affect the properties and activity of Novaferon protein or parts thereof. Conservative substitutions are also particularly preferred in this regard.

According to one aspect of the invention, there is provided an isolated nucleic acid molecule comprising a polynucleotide having a nucleotide sequence at least 93% identical and more preferably at least about 95%, 96%, 97%, 98% or 99% identical to the polynucleotide selected from the group consisting of: (a) a nucleotide sequence encoding a Novaferon protein having the complete amino acid sequence in SEQ ID No: 2 (i.e., positions 1-166 of SEQ ID No: 2); and (b) a nucleotide sequence encoding a biologically active fragment of the protein from (a); and (c) a nucleotide sequence complementary to any of the nucleotide sequences in (a) or (b) above.

Due to the degeneracy of the genetic code, it will immediately be apparent to one of ordinary skill in the art that a large number of nucleic acid molecules having a sequence of at least about 93%, 95%, 96%, 97%, 98%, or 99% identical to nucleic acid sequences from the nucleic acid sequence shown in FIG. 1 (SEQ ID No: 1), will encode a protein having an activity similar or identical to the Novaferon protein. In fact, since all degenerate variants encode the same protein, this fact will be obvious to a specialist even without a comparative analysis. It will also be apparent in the art that among such nucleic acid molecules that are not degenerate variants, a reasonable amount will also encode a protein having interferon-like biological activities. This is because the probability of amino acid substitutions with respect to a significant effect on protein function (for example, replacing one aliphatic amino acid with another aliphatic amino acid) is either small or non-existent, as is further described below, and the specialist is fully aware of this. For example, guidance on how to make phenotypically silent amino acid substitutions was proposed by Bowie et al. (70), where the authors show that many proteins are tolerant to amino acid substitutions.

Protein and polypeptide variants and constructs

The present invention encompasses Novaferon protein with SEQ ID No: 2 and protein or polypeptide variants substantially similar thereto, such as non-naturally occurring proteins having at least about 85-95% or more amino acid sequence identity with respect to SEQ ID No : 2. For example, non-naturally occurring proteins having at least about 85%, 90%, 95%, 96%, 97%, 98% or 99% sequence identity with respect to the amino acid sequence shown in SEQ ID No: 2 are within the scope of the invention . In addition, the Novaferon protein of the invention can be structurally modified by fusing it with other proteins or protein fragments or other molecules in order to enhance its functions and properties. Examples include, but are not limited to, fusing it to other proteins / protein fragments in order to increase expression or to further stabilize Novaferon protein.

In one embodiment, the Novaferon-coding nucleic acid sequence and / or Novaferon proteins of the invention may be labeled with a different label from the backbone. "Labeled" in this description means that the structure of the nucleic acid sequence (SEQ ID No: 1) or Novaferon protein (SEQ ID No: 2) is connected to at least one element, isotope or other chemical reagents (labels) in order to make possible detection of this structure. In general, labels are divided into three classes: a) isotopic labels, which can be radioactive or heavy isotopes; b) immune labels, which may be antibodies or antigens; and c) color or fluorescent dyes. Labels can be entered into the structure at any position.

After receiving the protein Novaferon can also be covalently modified. One type of covalent modification involves treating Novaferon protein with an organic derivative agent capable of interacting with selected side chains or N- or C-terminal residues of Novaferon protein. Derivation using bifunctional agents is useful, for example, for cross-linking Novaferon protein with a water-insoluble matrix or surface of a carrier for use in anti-Novaferon antibody purification or screening assays. Commonly used cross-linking agents include 1,1-bis (diazoacetyl) -2-phenylethane, glutaraldehyde, N-hydroxysuccinimide esters (e.g., 4-azidosalicylic acid esters), homobifunctional imido esters, including disuccinimidyl esters, such as 3.3 '-dithiobis (succinimidyl propionate), bifunctional maleimides, such as bis-N-maleimido-1,8-octane, and agents such as methyl-3 - [(p-azidophenyl) dithio] propioimidate.

Other modifications of the Novaferon protein include the deamidation of glutaminyl and asparaginyl residues to the corresponding glutamyl and aspartyl residues, respectively; hydroxylation of proline and lysine; phosphorylation of hydroxyl groups of seryl or threonyl residues; methylation of the amino groups of the side chains of lysine, arginine, and histidine (71); acetylation of the N-terminal amine and amidation of any C-terminal carboxyl group.

Another type of covalent modification of the Novaferon protein of the present invention involves altering the native glycosylation pattern of a given protein. This can be achieved, for example, by (1) delegating and / or adding one or more carbohydrate moieties found in the native sequence of the Novaferon protein, or (2) adding and / or deleting one or more glycosylation sites that are not in the native sequence of the Novaferon protein .

Addition of glycosylation sites to the Novaferon protein can be achieved by changing the amino acid sequence of the Novaferon protein. This change can be made, for example, by adding one or more serine or threonine residues or by replacing one or more serine or threonine residues in the native sequence of the Novaferon protein (for O-glycosylation sites). Such a change in the amino acid sequence of the Novaferon protein can be achieved by changes at the DNA level, in particular by introducing mutations into the DNA sequence encoding the Novaferon protein at preselected nucleotide bases so that the altered codons are translated into the desired amino acids.

Another way to increase the amount of carbohydrate moieties on the Novaferon protein is by chemical or enzymatic binding of glycosides to the protein. Such methods are described in the art, for example, already in 1981, Aplin JD and Wriston JC Jr. described the preparation, properties, and applications of carbohydrate conjugates of proteins and lipids (72).

Removal of carbohydrate moieties present on the Novaferon protein can be achieved chemically or enzymatically or by mutationally replacing codons encoding amino acid residues that serve as targets for glycosylation. Chemical deglycosylation techniques are known in the art and are described, for example, by Edge AS et al. (73). Enzymatic cleavage of carbohydrate moieties from polypeptides can be achieved by using a number of endo- and exoglycosidases, as described by Thotakura et al. (74).

Such derivatized constructs may include groups that improve solubility, absorption, penetration through the blood-brain barrier, biological half-life, etc. Thanks to such groupings or modifications of Novaferon protein, it is possible to alternatively eliminate or reduce any possible unwanted side effects of the protein and the like. Groups capable of mediating such effects are described, for example, in Remington: The Science and Practice of Pharmacy (75).

Another type of covalent modification of Novaferon involves binding of Novaferon protein to one of a number of non-protein polymers, for example polyethylene glycol, polypropylene glycol or polyoxyalkylenes, for example, by the method described in US patent No. 4640835 (76), 4496689 (77), 4791192 (78) or 4179337 ( 79).

In addition, the Novaferon protein of the present invention can also be modified by the method to form chimeric molecules containing Novaferon protein fused to another heterologous polypeptide or amino acid sequence. In one embodiment, such a chimeric molecule contains a fused structure of a Novaferon protein with a labeled polypeptide, thereby providing an epitope to which an anti-tag antibody can selectively bind. An epitope tag is usually placed at the amino or carboxyl end of Novaferon protein. The presence of such forms of the Novaferon protein with a labeled epitope can be detected using an anti-tag polypeptide antibody. In addition, the provision of an epitope tag makes it possible to easily purify Novaferon protein by affinity purification using antibodies against the tag or a different type of affinity matrix that binds to the tag epitope. In an alternative embodiment, the chimeric molecule may comprise a fused structure of a Novaferon protein with an immunoglobulin or a specific immunoglobulin site / fragment. For example, to form a bivalent form of a chimeric molecule, Novaferon protein can be fused to the Fc region of an IgG molecule.

Various tag polypeptides and their corresponding antibodies are well known in the art. Examples include poly-histidine (poly-hist) or poly-histidine-glycine (poly-gly-gly) labels; the flu-HA polypeptide tag (influenza virus hemagglutinin) and its 12CA5 antibody (80); label c-myc and 8F9, 3C7, 6E10, G4, B7 and 9E10 and antibodies to it (81); and the herpes simplex virus glycoprotein D (gD) label and antibody thereto (82). Other tag polypeptides include the Flag peptide (83); the tubulin epitope peptide (84) and the peptide tag of the 10 T7 gene protein (85).

In addition, the Novaferon protein of the present invention can be obtained by chemical synthesis techniques known to those of ordinary skill in the art. For example, polypeptides up to and including 80-90 amino acid residues in length can be prepared using a commercially available peptide synthesizer, model 433A (Applied Biosystems, Inc., Foster City, CA, US). In addition, longer chemically synthesized peptides of up to 120 residues are also commercially available, for example, from Bio-syhthesis, Inc., Lewisville, TX, USA. Thus, as you can easily understand, the full-sized mature protein Novaferon can be obtained synthetically (for example, in the form of fragments, which can then be assembled together).

Therefore, the Novaferon protein of the present invention (SEQ ID No: 2) includes all protein and polypeptide compositions and constructs having the same amino acid sequence as described in SEQ ID No: 2, regardless of whether these Novaferon proteins and protein derivatives using chemical synthesis techniques and / or by recombinant techniques from prokaryotic or eukaryotic host cells or other cells and hosts, including, but not limited to, bacteria, yeast, plants, insects and mammalian cells . Depending on the hosts used in the recombinant production method, the proteins of the present invention can be glycosylated or non-glycosylated, pegylated or non-pegylated. In addition, the proteins of the invention may also include, in some cases, an initially modified methionine residue as a result of host-mediated processes. Thus, it is well known in the art that the N-terminal methionine encoded by the translation initiation codon is usually removed with high efficiency from all proteins after translation in all eukaryotic cells. Although the N-terminal methionine of most proteins is also effectively removed in most prokaryotes, for some proteins this prokaryotic removal process is ineffective depending on the nature of the amino acid to which the N-terminal methionine is covalently linked.

Getting

The present invention also relates to recombinant vectors that consist of the isolated DNA molecules of the present invention, to host cells that are genetically constructed / transfected using these recombinant vectors, and to the preparation of Novaferon protein or fragments thereof by recombinant techniques. The vector may be, for example, a plasmid, phage, viral or retroviral vector. Retroviral vectors may be replication competent or replication defective. In the latter case, virus propagation will usually occur only in complementary host cells. Examples detailing the preparation of Novaferon are given below.

Preferred vectors for the expression of the Novaferon protein of the present invention include, but are not limited to, vectors containing cis-acting control regions effective for expression in the host, operably linked to a polynucleotide for its expression. Corresponding trans-acting factors are delivered either by the host via a complementing vector, or by the vector itself after introduction into the host.

The nucleic acid sequence described in the present invention (SEQ ID No: 1) can be operatively linked to the corresponding promoter. "Promoter" as used herein means any nucleic acid sequence capable of binding to RNA polymerase and to initiate the extron (usually in the forward direction (3 ')) transcription of the coding sequence for the Novaferon protein in mRNA. The bacterial promoter has a transcription initiation site, which is usually located proximal to the 5'-end of the coding sequence. This transcription initiation site typically includes an RNA polymerase binding site and a transcription initiation site. Sequences encoding metabolic pathway enzymes provide particularly useful promoter sequences. Examples include promoter sequences for sugar metabolism enzymes such as galactose, lactose and maltose, and sequences for biosynthesis enzymes such as tryptophan. Bacteriophage promoters may also be used, and they are known in the art. In addition to this, synthetic promoters and hybrid promoters are also useful; for example, the tac promoter is a hybrid of the trp and lac promoter sequences. In addition, the bacterial promoter may include naturally occurring promoters of non-bacterial origin that have the ability to bind to bacterial RNA polymerase and initiate transcription. Preferred bacterial promoters include, but are not limited to, the E. coli laci, trp, phoA and lacZ, T3 and T7 promoters, the gpt promoter, lambda PR, PL promoters, and the trp promoter.

Eukaryotic promoters have a transcription initiation site, which is usually located proximal to the 5'-end of the coding sequence, and a TATA box, usually located at a distance of 25-30 base pairs (bp) upstream from the site of transcription initiation. It is believed that the TATA box directs RNA polymerase II to the start of RNA synthesis from the correct site. The mammalian promoter also contains an upstream promoter element (enhancer element), typically located within 100-200 base pairs upstream from the TATA box. The upstream promoter element determines the rate at which transcription is initiated and can act in either of two orientations. The mammalian promoters are specifically used as mammalian promoters, since viral genes are often highly expressed and have a wide host range. Examples include the SV40 early promoter (monkey virus 40), the viral LTR promoter of a mouse breast tumor. Preferred animal cell promoters include, but are not limited to, the adenovirus major late promoter, the herpes simplex virus promoter, and the CMV (cytomegalovirus) promoter. Among the known eukaryotic promoters, the CMV immediate early promoter, the elongation factor 1 alpha (EF1A) promoter, the thymidine kinase promoter HSV (herpes simplex virus), the early and late SV40 promoters, and the retroviral LTR promoters, such as those from Routh sarcoma virus ( "RSV"). Preferred promoter sequences for expression in yeast include the inducible promoter GAL1 / 10, promoters of the gene for alcohol dehydrogenase, enolase, glucokinase, glucose-6-phosphate isomerase, glyceraldehyde-3-phosphate dehydrogenase, hexokinase, phosphofructokinase, 3-phosphofructokinase, 3-phosphofructokinase, 3-phosphate kinase, 3-phosphate kinase. acid phosphatase.

In addition, propagation and expression vectors typically include one or more selectable markers. Such markers may be suitable for amplification or the vectors may contain additional markers for this purpose. In this regard, expression vectors preferably contain one or more selectable marker genes in order to provide a phenotypic feature for the selection of transfected host cells, although those skilled in the art will recognize that some selectable markers in systems can be represented on separate vectors. Preferred markers include, for example, ampicillin (Amp), tetracycline (Tet) or hygromycin (HYG) resistance genes for cultivation in E. coli and other bacteria. Yeast selectable markers include ADE2, HIS4, LEU2, TRP1 and ALG7, which confer tunicamycin resistance; the neomycin phosphotransferase gene, which confers resistance to G418; and the CUP1 gene, which allows yeast to grow in the presence of copper ions. Markers that are selectable in animal cells include the dihydrofolate reductase (DHFR) gene, neomycin resistance (Neo) or hygromycin resistance (HYG) genes.

In addition, the vectors for propagation and expression usually contain one or more sites of initiation, termination, transcription and, in the transcribed region, a binding site with a ribosome for translation. The coding portion of the transcripts expressed by these constructs preferably includes a translation initiation codon at the beginning and a termination codon (UAA, UGA or UAG), respectively located at the end of the translated DNA sequence. The choice of promoters, terminators, selectable markers, vectors, and other elements is a matter of generally accepted construction corresponding to the level of ordinary skill in the art. Many of these elements are described in the literature and are available from commercial suppliers.

The following vectors are commercially available and are preferred for use in bacteria: pBV220 (86) and its derivatives from Shanghai Sangon; pQE species from Qiagen; pET vectors from Qiagen; pBS vectors, Phagescript vectors, Bluescript vectors, pNH8A, pNH16a, pNH18A, pNH46A from Stratagene; and ptrc99a, pKK223-3, pKK233-3, pDR540, pRIT5 from Pharmacia. Among the preferred eukaryotic vectors are pCI vectors from Promega, pcDNA vectors from Invitrogen, pSV2CAT, pOG44, pXT1 and pSG from Stratagene; and pSVK3, pBPV, pMSG and pSVL from Pharmacia. These vectors are listed solely as examples to demonstrate that many commercially available and well-known vectors are available to those skilled in the art for use in the production of the Novaferon protein described in the present invention by genetic / recombinant methods.

In some preferred embodiments, in this sense, the vectors provide means for specific expression. Such specific expression can be inducible expression or expression only in certain types of cells, or it can be both inducible and cell-specific. Particularly preferred among inducible vectors are vectors that can be induced for expression by environmental factors that are easy to manipulate, such as temperature and nutritional supplements. A number of vectors suitable for this application, including constitutive and inducible expression vectors for use in prokaryotic and eukaryotic hosts, are well known and regularly used by specialists in this field of technology.

A vector containing the DNA sequence described in SEQ ID No: 1, for example, as well as the corresponding promoter and other appropriate control sequences can be introduced using a number of techniques known in the art into an appropriate host cell suitable for expression of the desired protein. Representative examples of such suitable hosts include bacterial cells such as E. coli, Bacillus subtilis, Streptomyces cells; yeast cells, such as Pichia pastoris cells; insect cells such as Drosophila S2 and Spodoptera Sf9 cells; mammalian cells, such as CHO (Chinese hamster ovary) and COS (African green monkey fibroblasts); and plant cells. The hosts for a number of expression constructs are well known, and those skilled in the art will be able, using the information described in the present invention, to easily select a host for expression of the Novaferon protein described in SEQ ID No: 2.

Host cells can be genetically engineered to incorporate the Novaferon-coding polynucleotides and expression of the Novaferon proteins of the present invention. For example, Novaferon-coding polynucleotides can be introduced into host cells using transfection techniques known in the art. Such methods are described in many standard laboratory manuals, such as those reviewed by Kingston (87). Novaferon-coding polynucleotides can be introduced / transfected on their own or with other polynucleotides. Such other polynucleotides may be independently introduced, co-administered, or simultaneously administered with the Novapheron-coding polynucleotides described in SEQ ID No: 1.

For example, the Novaferon-encoding polynucleotides of the invention can be transfected into host cells along with a single polynucleotide encoding a selectable marker for co-transfection and selection of the marker in mammalian cells. Alternatively, Novaferon-encoding polynucleotides can be included in a vector containing a selectable marker-encoding DNA sequence to induce propagation in host cells.

Constructed host cells transfected using vectors containing Novaferon-coding polynucleotide can be cultured in traditional culture media that can be specifically modified to activate promoters, select transformants, or amplify target genes. Cultivation conditions, such as temperature, pH, etc., that are suitable for the selected host cells for the expression of the Novaferon protein of the present invention are selected.

Suitable secretion signals can be incorporated, and they can be co-expressed with Novaferon protein to stimulate the secretion of the translated protein polypeptide into the lumen of the endoplasmic reticulum, into the periplasmic space, or into the extracellular environment.

Cleaning

For the expression of the target recombinant protein, the appropriate type of host cells is usually selected depending on the nature of the target protein and other conditions, such as the cost of production, the availability of easy scaling, the volume of industrial production, etc. Then, clones of transfected cells expressing the target protein with the highest yield are selected, the final clone with optimal expression is called the cell line expressing the target protein and used to obtain the target protein. The cell line expressing the target protein is grown in a medium containing various nutrients. For the purpose of optimal cell growth and / or optimal expression of the target protein, various agents or conditions are used to induce a selective promoter incorporated with the cDNA sequence of the target protein into a transfection vector. If the host cell type / expression system is bacteria, cultured cells are harvested from this medium, usually by centrifugation. The main part of the collected cells is destroyed by physical or chemical methods and the collected crude extracts containing the synthesized target protein are stored for further protein purification. Methods useful for disrupting microbial cells include, but are not limited to, freeze-thaw cyclic treatment, ultrasonic irradiation, mechanical disruption, or the use of cell lysing agents. Such methods are well known to those skilled in the art.

The inventors used E. coli bacteria as a host cell for expression of the recombinant Novaferon protein. As described below, E. coli was transfected using a vector that contained the Novaferon-coding polynucleotide sequence, and one E.coli strain that had optimal expression of the Novaferon protein was selected to produce the Novaferon protein. Once synthesized, the protein can be stored in the cytoplasm as insoluble granules or can be secreted into the cytoplasm in soluble form. In the first case, the granules are removed after lysis of the inclusion bodies and denatured using, for example, guanidine isothiocyanate or urea. The refolding of the denatured Novaferon polypeptide / protein is further achieved by diluting the denaturing agent with an excess of diluting solution or dialyzing against a urea solution and a combination of reduced and oxidized glutathione followed by dialysis against buffered saline. In the second case, the protein can be directly extracted without a denaturing agent from the periplasmic space in a soluble and functional form after the destruction of the collected cells. The soluble Novaferon protein, which avoided the use of denaturation and refolding, is intact and does not contain deformed or improperly folded protein molecules.

The inventors found that a significant portion of the synthesized Novaferon protein obtained in the E. coli cell line was secreted into the cytoplasm. This portion was then purified as described below.

Activity analyzes and medical applications

As indicated above, Novaferon protein shows sequence homology with many members of the interferon family, in particular with the interferon protein translated from HuIFN-α14 mRNA (FIG. 2). HuIFN-α has been shown to have a wide range of biological activities, including antiviral, antiproliferative, and immunomodulatory activities (10).

With this homology with HuIFN-α, Novaferon can be expected to exhibit biological functions similar to HuIFN-α, including, but not limited to, inhibition of tumor proliferation, antiviral activity, activation of NK cells and modulation of the immune system. Of particular importance is not only the preservation of HuIFN-α-like functional properties, but also the increased effectiveness of these biological functions of the Novaferon protein compared to HuIFN-α. Thus, to test and determine the effectiveness of its functional properties, the biological activities of Novaferon protein were tested using classical and generally accepted in vitro assays designed to detect antiviral and antiproliferative properties. As described in the experimental section below, the in vivo efficacy of the antiproliferative properties of Novaferon protein was further observed in animal models of various types of human cancer and compared with HuIFN-a, as well as with a chemical anticancer agent in some experiments.

A large number of suitable assays for determining HuIFN activity are well known in the art. The inventors used in vitro cell assay systems to determine antiviral and antiproliferative activities. The same in vitro assays were used for all methods and experiments related to the present invention, which included, but were not limited to, screening of the shuffling library of human interferon genes, type I, selection of Novaferon from the expressed proteins of the shuffling library of human interferon genes, type I, and determination of the biological activities of the pure recombinant protein Novaferon.

There are many assays that measure the antiviral activity of test samples / agents by monitoring the degree of cell resistance to viruses (88). Three principal biological assays were used to measure the antiviral activities of HuIFN and their hybrids. They are classified according to methods for determining various aspects of the virus on cultured cells.

In the assay for determining the inhibition of virus-induced cytopathic effects, the degree of reduction of the virus-induced lytic cytopathic effects on cultured cells with IFN pretreatment is measured. This analysis can be performed in 96-well plates (89), and it is widely used for recombinant HuIFN-α because it provides a simple method for screening a large number of samples.

Inhibition of viral plaque formation is another method for quantifying the antiviral activity of HuIFN in tissue cultures. The results of the analysis of reducing the number of plaques do not depend on the multiplicity of infection. Moreover, a 50% reduction in plaque formation is measured with high accuracy. The use of the ubiquitous vesicular stomatitis virus (VSV) to induce plaque formation, for example, makes it possible to determine the cross-species activity profile of a specific recombinant IFN by screening a number of cell lines of various animal species (90).

The third analysis is based on determining the decrease in virus output. Typically, virus production is measured over a single cell growth cycle by the amount of virus released. This assay is particularly useful for testing antiviral IFN activities against viruses that do not cause cytopathic effects or that do not form plaques in target cell cultures. In this test, however, the multiplicity of infection affects the apparent degree of protection caused by a fixed concentration of IFN (91).

The antiviral activities of Novaferon were measured using a standard cytopathic inhibition assay using WISH cells and vesicular stomatitis virus (VSV). Antiviral activities were determined and calibrated using standard reference samples of international standards BO3: 95/650 (rHuIFN-α2a) and 94/786 (rHuIFN-α consensus). One unit of antiviral activity is defined as the amount of protein required to achieve 50% inhibition of the cytopathic effects of VSV on cultured cells. As further described below, the activity of Novaferon protein was 2.5 × 10 ⁹ IU / mg, which is approximately 12.5 times greater than the activity of HuIFN-α2b. These tests demonstrate that the antiviral properties of Novaferon are significantly improved compared to HuIFN-α2b. This enhanced virus efficacy exhibited by Novaferon provides the basis for the alleged improved in vivo antiviral effects in humans. Based on the nature of HuIFN, it is reasonable to expect Novaferon to have a very broad antiviral profile. In other words, Novaferon should be more effective against a wide range of viruses than natural HuIFN. The increased antiviral efficacy of Novaferon can be expressed in the form of improved antiviral effects or improved therapeutic effects in the clinical setting for patients with various viral diseases.

As explained above, IFNs also inhibit cell proliferation and have powerful antitumor effects using a number of mechanisms. Several in vitro antiproliferative assays using cell culture systems have been approved and well described in the art. Cell proliferation in these assays can be measured by counting the number of cells; biological analysis using crystalline violet (92, 93); chemical sensitivity to the neutral red dye (94-96); inclusion of radioactive labeled nucleotides (97); incorporation of 5-bromo-2'-deoxyuridine (BrdU) into the DNA of proliferating cells (98); the use of tetrazolium salts (99, 100).

The Daudi human lymphoblastoid cell line is very sensitive to the antiproliferative effect of HuIFN-α, and its growth in suspension cultures facilitates the determination of the number of its cells (101). This cell line has been used to measure the antiproliferative activity of HuIFN-α and hybrids for many years (102). Other cell lines are also used to test the antiproliferative activity of the test agent.

The antiproliferative activity of Novaferon protein was observed in vivo, tracking the inhibition of tumor mass growth by the introduction of Novaferon in animal models with different xenografts of human tumors. The in vivo antitumor effects of Novaferon were compared with HuIFN-α2b and, in some xenograft models, also with a chemical antitumor agent.

As described in more detail below, the inventors found that the in vitro antiproliferative activity of Novaferon, measured using the standard method with Daudi cells, was 400 times stronger than that of natural HuIFN-α2b, which demonstrates probably the most potent antiproliferative activity of all natural HuIFN . The increased antiproliferative efficacy of Novaferon was broad and universal in nature, since it showed more effective and increased inhibition than natural HuIFN-α2b for all human cancer cell lines tested in vitro by the inventors. This indicates that effective inhibition of human cancer by Novaferon is not selective. Despite the fact that the degree of its increased antiproliferative activity with respect to all tested types of human cancer cell lines varied, Novaferon has the potential of an anticancer agent of wide action in clinical conditions. This represents a significant advantage over chemical anticancer agents, monoclonal antibodies and other anticancer agents directed at specific targets.

In experiments on animal models with xenografts described below, it was further established that:

(1) the in vivo antiproliferative effects of Novaferon were significantly enhanced or more effective than natural HuIFN-α2b;

(2) the in vivo antiproliferative effects of Novaferon at lower doses were better than the test chemical agent 5-fluorouracil (5-FU) in the same xenograft model;

(3) the use of Novaferon made it possible to achieve more than 90% inhibition of cancer growth in xenograft models, however, it did not induce weight reduction, changes in activity and other negative side effects in the treated animals, which contrasted sharply with a significant decrease in weight and a decrease in activity in 5- FU-treated animals.

These results show that the antiproliferative properties of in vitro and in vivo Novaferon are significantly enhanced compared with natural HuIFN-α2b. The increased antiproliferative efficacy of Novaferon is expressed in the effective inhibition (more than 90%) of human tumor growth in the mouse animal model, and this inhibition appears to act over a very wide range for all types of human cancer tested and better than the classic chemical anti-cancer agent 5FU . These results also show that effective inhibition of cancer cell growth by Novaferon is very specific to the cancer cell, but not to normal cells, as evidenced by the observation of normal feed intake and active behavior and the absence of any weight reduction in Novaferon-treated animals. Therefore, Novaferon has the potential to affect all or most types of human cancer.

In a preferred embodiment, the full-sized molecule (s) or incomplete molecule (s) of Novaferon protein (SEQ ID No: 2) obtained by recombinant methods using the polynucleotide sequence of SEQ ID No. 1 or chemically synthesized, could be used to treat and / or prevent any and / or all tumors and types of cancer of human origin or non-human origin in humans and / or non-human species. These tumors, for example, include, but are not limited to, osteogenic sarcoma; multiple myeloma; Hodgkin's disease; weakly differentiable nodular lymphoma; acute lymphocytic leukemia; acute myeloid leukemia; mammary cancer; melanoma; papilloma; cancer of the nasopharyngeal region, colon cancer, liver cancer and melanoma.

In another embodiment, the full-sized molecule (s) or incomplete molecule (s) of Novaferon protein (SEQ ID No: 2), obtained by recombinant methods using the polynucleotide sequence of SEQ ID No: 1 or chemically synthesized, could be used for treatment and / or prevention any and / or all viral diseases in humans and / or non-human species. Examples of susceptible viral infections include, but are not limited to, viral encephalomyocarditis, influenza and other viral infections of the respiratory tract, rabies and other viral zoonoses, and arbovirus infections, as well as herpetic keratitis, acute hemorrhagic conjunctivitis, chickenpox plus shingles (varicella z) and hepatitis B and C, SARS (SARS) and bird flu, human immunodeficiency syndrome (AIDS, HIV).

In another embodiment, the full-sized molecule (s) or incomplete molecule (s) of Novaferon protein (SEQ ID No: 2), obtained by recombinant methods using the polynucleotide sequence of SEQ ID No: 1 or chemically synthesized, could be used for treatment and / or prevention any and / or all disorders of the immune system in humans. Examples of immune disorders include, but are not limited to, rheumatoid arthritis, multiple sclerosis, and diabetes in Sjogren's syndrome. In addition, Novaferon protein can also be used to prevent graft versus host rejection.

In another embodiment, the full-sized molecule (s) or incomplete molecule (s) of Novaferon protein (SEQ ID No: 2), obtained by recombinant methods using the polynucleotide sequence of SEQ ID No: 1 or chemically synthesized, could be used for treatment and / or prevention , as an immunoadjuvant, of any and / or all angiogenesis diseases. Examples of angiogenesis diseases include, but are not limited to, hemangiomas, tumor-induced neovascularization, age-related macular degeneration, and diabetic retinopathy.

Novaferon protein, alone or in combination with any other carrier proteins / substances or other constructs, can be administered to humans and / or non-human species in any pharmaceutically acceptable formulations / compositions by any means of administration / delivery routes that include, but are not limited to , oral administration, inhalation, intranasal nebulization, intraperitoneal, intravenous, intramuscular injection, intravenous injection, or subcutaneous injection.

Pharmaceutical preparations / compositions containing Novaferon protein as an active ingredient could be made by incorporating an appropriate solid or liquid carrier, in the form of a liquid, solid, semi-solid and / or any other clinically acceptable form, such as tablets, pills, powders , liquid solutions or suspensions, liposomes, suppositories, solutions for injection and infusion. Novaferon-containing preparations / compositions could be made using traditional carriers, substances, methods described in the art or commonly accepted by the pharmaceutical industry. Novaferon-containing preparations / compositions could also be made using unconventional methods, substances that are not described in the art and are also not used by the pharmaceutical industry.

For example, parenteral compositions are usually injectable fluids consisting of pharmaceutically and physiologically acceptable substances, such as water, saline, other balanced saline solutions, an aqueous solution of dextrose, glycerol, etc. In addition, the fluids introduced by injection could also contain, in addition to the Novaferon protein, other proteins as carriers, such as human serum albumin or plasma preparations. Pharmaceutical preparations / compositions may also contain minor amounts of non-toxic adjuvants, such as wetting or emulsifying agents, preservatives, and pH-maintaining buffering agents (e.g., sodium acetate or sorbitan monolaurate). Methods for making the compositions are well known in the art and are described, for example, in Remington: The Science and Practice of Pharmacy. Phamaceutical Sciences (75).

Specific preparations / compositions based on Novaferon protein will be determined in accordance with the planned clinical applications and / or methods of administration, and they could be made by a person skilled in the art using known techniques. For example, in addition to injectable fluids, topical and oral compositions may be employed. Topical preparations may include, but are not limited to, eye drops, ointments, and sprays. Oral compositions include, but are not limited to, forms of liquids (eg, syrups, solutions or suspensions) or solids (eg, powders, pills, tablets or capsules). For solid preparations / compositions, conventional non-toxic solid carriers include, but are not limited to, pharmaceutical grades of mannitol, lactose, starch or magnesium stearate. Actual techniques and / or methods for the manufacture of these preparations / compositions are known or will be apparent to those skilled in the art (75).

Pharmaceutically acceptable Novaferon protein preparations / compositions can be administered to humans and / or non-human species in a number of ways that include, but are not limited to, oral, subcutaneous, intravenous, intranasal, transdermal, intraperitoneal, intramuscular, intrapulmonary, vaginal, rectal or intraocular delivery, and in the treatment of wounds by direct local application.

The concentration / amount of Novaferon protein in the preparations / compositions may vary from more than 0 to 1.0 molar and / or from more than 0 to 100% (w / w) depending on clinical practice. The exact doses, intervals of administration and duration of treatment with each and / or all Novaferon drugs / compositions will be determined by clinical trials, disease states, patient status and health care providers. In a preferred embodiment, due to protein degradation, systemic, compared to localized, delivery and synthesis rate of new proteases, as well as age, body weight, general health, sex, nutrition, time of administration, drug interaction, severity, etc. adjustments for the administration of Novaferon may be necessary, including, but not limited to, individual and / or total doses, administration intervals, duration of treatment, and necessary courses of treatment, and they will be refined using routine experimentation by those skilled in the art.

In a preferred embodiment, the half-life of Novaferon protein from the bloodstream after its introduction into humans and / or non-human species may change. Changes include, but are not limited to, lengthening or shortening the half-life of Novaferon in vivo. The extension of the half-life of Novaferon protein in vivo can be achieved in various ways, which include, but are not limited to:

(1) the formation of a complex between the Novaferon molecule and a monoclonal antibody. Such an antibody will preferably be associated with the Novaferon protein at sites whose connection does not fundamentally impair its therapeutic function (103);

(2) the formation of a fusion complex of Novaferon with other proteins / polypeptides. The Novaferon molecule can be recombinantly fused with other proteins / polypeptides, such as a fragment of an immunoglobulin (Fc) constant region (104);

(3) conjugation of Novaferon protein. For example, Novaferon protein can be conjugated to non-antigenic polymers such as polyethylene glycol or related polyalkylene glycol groups (105-108).

In another preferred embodiment, a therapeutic compound could be conjugated to an antibody, preferably an anti-Novaferon protein antibody. The therapeutic compound may be a cytotoxic agent. In this method, cytotoxic agents can be targeted, by binding the conjugated antibody to Novaferon molecules, to tumor tissue or cells, thereby destroying and reducing the number of affected cells to achieve a reduction in cancer symptoms. Cytotoxic agents include, but are not limited to, cytotoxic drugs, toxins, or active fragments of such toxins and radioactive chemicals. Suitable tkosins and their corresponding fragments include diphtheria toxin chain A, exotoxin chain A, ricin chain A, abrin chain A, curcin, crotin, phenomycin, enomycin and the like.

In a preferred embodiment, the full-length sequence, incomplete sequences and / or regulatory sequence of the Novaferon protein-coding polynucleotide sequence (SEQ ID No: 1) can be used for gene therapy by any person skilled in the art. The use of antisense sequences based on the Novaferon protein-encoding polynucleotide sequence (SEQ ID No: 1) can also be used either as gene therapy (i.e., for inclusion in the genome) or as antisense compositions, as will be apparent to those skilled in the art .

As for gene therapy applications, genes are introduced into cells to achieve in vivo synthesis of target proteins encoded by these genes. Conventional gene therapy achieves sustained therapeutic effects as a result of a single treatment or repeated administration of a therapeutically effective DNA or mRNA. On the other hand, antisense RNAs and DNA can also be used as therapeutic agents to block the expression of certain genes in vivo. It has already been shown that short antisense oligonucleotides can be delivered to cells, where they act as inhibitors (109).

In a preferred embodiment, the Novaferon protein-coding polynucleotide sequence (SEQ ID No: 1), full-length or incomplete length, can be used as DNA vaccines. Generally, naked DNA vaccines are known in the art (110). Methods of using the Novaferon coding gene (SEQ ID No: 1), full-size or incomplete length, as DNA vaccines are well known to those of ordinary skill in the art and include, but are not limited to, placing the Novaferon gene or part of the Novaferon gene under control a promoter for the expression of the full-length Novaferon protein or Novaferon protein of incomplete length in humans and / or non-human species.

Examples

The following examples serve to more fully represent the method of application of the invention described above, as well as to present the best methods contemplated for implementing various aspects of the invention. It is understood that these examples in no way limit the true scope of this invention, but rather are presented for illustrative purposes. All references cited herein are expressly incorporated in their entirety by reference.

Example 1. PCR amplification of human IFN-α genes from human leukocyte cDNA

Total mRNA was extracted from human peripheral blood leukocytes. Obtaining cDNA was performed using the Advantage ™ RT-for-PCR kit (Clontech, Mountain View, CA, US) and synthetic cDNA primers (oligo-dT18) in accordance with the manufacturer's recommendations.

Amplification of human IFN-α cDNA was carried out using PCR technology on a MJ PTC thermocycler using the following conditions: 2.5 μl of 10x pfx amplification buffer (Invitrogen, Carlsbad, CA, US); 0.75 μl of 10 mM dNTP (decoxynucleotide triphosphates); 0.5 μl of 25 mM MgSO ₄ ; 0.25 μl Platinum pfx DNA polymerase (2.5 U / μl; Invitrogen, Carlsbad, CA, US); 0.75 μl of cDNA; 0.75 μl of the 5'-primer (10 μM; IFNaO5: 5'-TGGTGCTCAGCT (A / G) CAAGTC-3 '), (SEQ ID No: 3); 0.75 μl of a mixture of 3'-primers (1.7 μm each:

IFNaO3-1: 5'-AATCATTTCCATGTTG (A / G) ACCAG-3 '(SEQ ID No: 4);

IFNaO3-2: 5'-AATCATTTCCCGGTTGTACCAG-3 '(SEQ ID No: 5);

IFNaO3-3: 5'-AATCATTTCCATGTTGAAACAG-3 '(SEQ ID No: 6);

IFNaO3-4: 5'-AATCATTTCAAGATGAGCCCAG-3 '(SEQ ID No: 7);

IFNaO3-5: 5'-AATGATnTCATGTTGAACCAG-3 '(SEQ ID No: 8);

IFNaO3-6: 5'-AATCATTT (C / G) (C / G) ATGTTGAACCAG-3 '(SEQ ID No: 9);

IFNaO3-7: 5'-GATCATTTCCATGTTGAATGAG-3 '(SEQ ID No: 10);

IFNaO3-8: 5'-GAGTCGTTTCTGTGTTGGATCAG-3 '(SEQ ID No: 11).

The amplified PCR products were subjected to electrophoresis on a 1.0% agarose gel, excised, gel purified and cloned into the pCRII-TOPO or pCR-4-TOPO vector (Invitrogen, Carlsbad, CA, US) according to the manufacturer's recommendations. Automatic sequencing was performed on an ABI automated sequencer (PE Applied Biosystems, CA, US) using a Prism Ready Reaction Dye Termination mixture.

Since no desired inserts were found in the above clones for the coding sequences of IFNa6, IFNa7 and IFNa16, PCR was performed again under the conditions described above except for the type of specific primers. For specific amplification of IFNa6, 5'- and 3'-primers IFNaO5: 5'-TGGTGCTCAGCT (A / G) CAAGTC-3 '(SEQ ID No: 3) and IFNaO3-8: 5'-GAGTCGTTTCTGTGTTGGATCAG-3' (SEQ ID No: 11), respectively. For specific amplification of IFNa7, 5'- and 3'-primers IFNa7UO were used: 5'-ATGCCCCTGTCCTTTTTTTTAC-3 '(SEQ ID No: 12) and an equimolar mixture of IFNaO3-5 and IFNaO3-6, respectively. For specific amplification of IFNa16, the 5'- and 3'-primers IFNa7UO and IFNaO3-7: 5'-GATCATTTCCATGTTGAATGAG-3 '(SEQ ID No: 10), respectively, were used. The amplified fragments were cloned into the pCRII-TOPO or pCR-4-TOPO vector and sequenced as described above.

All cloned genes of human IFN-alpha, type I, were individually aligned with the corresponding DNA sequences from Genebank. For these referenced genes in this description, the nucleotide numbers in GeneBank were used: NMJ524013 (IFN-α1), NM_000605 (IFN-α2b), NM_010504 (IFN-α4), NM_010505 (IFN-α5), NM_008335 ( IFNα6), NM_008334 (IFN-α7), NMJ508336 (IFN-α8), NM_002171 (IFN-α10), NM_002172 (IFN-α14), NM_002173 (IFN-α16), NM_021268 (IFN-α17), NM_002175 (IFN-α21) )

Example 2. Construction of shuffling libraries of plasmids bearing HuIFN, type I

To construct plasmids carrying the coding sequence of one of the human IFN-α, type I, 15 pairs of oligonucleotides were synthesized, with restriction sites for BamHI and EcoRI (Genentech, South San Francisco, CA, US), based on the coding region of an individual cDNA for mature human IFN proteins, type I. For these genes that are referenced in this description, used the reference numbers for the nucleotides from GeneBank: NM_024013 (IFN-α1), NM_000605 (IFN-α2), NM_010504 (IFN-α4), NM_010505 (IFN -α5), NM_008335 (IFN-α6), NM_008334 (IFN-α7), NM_008336 (IFN-α8), NM_002171 (IFN-α10), NM_002172 (IFN-α14), NM_002173 (IFN-α16), NM_021268 (IFN- α17), NM_002175 (IFN- 21). Primers and plasmids designed in Example 1 as templates were used in standard PCR (111). The resulting products were digested with restriction endonucleases (REs) of BamHI and EcoRI, and the expression vector pBVB, which is a derivative of the expression plasmid pBV220 (86), containing the BamHI site and the EcoRI site in its multiple cloning region, was cloned into E. coli. All of these final constructs were verified by DNA sequencing analysis (PE Applied Biosystems, US).

DNA fragments containing OPC of human IFN were amplified by PCR using oligonucleotide pairs, BVF4: 5'-AGGGCAGCATTCAAAGCAG-3 '(SEQ ID No: 13) and BVR3: 5'-TCAGACCGCTTCTGCGTTCTG-3' (SEQ ID No: 14 and using previously constructed plasmids carrying HuIFN type I. The resulting products were mixed in equal amounts and digested with DNase I and PCR assembly in accordance with the procedure described by Stemmer (112).

The assembled PCR products were further amplified with a pair of internal primers: BVF: 5'-GAAGGCTTTGGGGTGTGTG-3 '(SEQ ID No: 15) and BVR: 5'-AATCTTCTCTCATCCGC-3' (SEQ ID No: 16), followed by digestion with Bam and Bam EcoRI and cloning back into E. coli pBVB expression vector cleaved using BamHI RE and EcoRI. All of these final constructs were verified by DNA sequencing analysis. Plasmids carrying the shuffled HuIFN-a genes transformed competent E. coli DH5α cells.

In all the above methods using PCR, or PCR amplification, or PCR assembly, conventional DNA polymerase (New England Biolab, MA, US) was used instead of high fidelity DNA polymerase.

Example 3. Screening of shuffling libraries

Freshly transformed E. coli DH5α cells were grown overnight on LB plates (Luria broth) at 37 ° C. Single colonies were individually selected and 100 μl of LB medium containing 50 μg / ml ampicillin were inoculated with them in 96-well plates. The colonies were shaken at 250 rpm at 30 ° C. At the end of overnight cultivation, 10 μl of bacterial cultures were inoculated in duplicate with 100 μl of LB medium containing 50 μg / ml ampicillin in 96-well plates. The original cups (so-called stock plates) were temporarily stored at 4 ° C. Cells on plates in duplicate were grown at 30 ° C. until an OD ₆₀₀ of 0.4 was achieved, and then induced at 42 ° C. After 4 hours of thermal induction, the bacterial cultures were directly transferred to the -80 ° C freezer in order to start the freeze-thaw cycle. After 2 freeze-thaw cycles, the bacterial suspension / lysate was diluted to the desired concentration and thereby affected the Daudi cell culture for antiproliferation test (101) or the Wish cell culture for antiviral test (113).

In each round of screening steps, 20,000 colonies were initially screened, and approximately 100 colonies with the highest antiproliferative or antiviral activity were selected for subsequent confirmatory testing. Selected bacterial cultures on stock plates were streaked onto LB plates containing 50 μg / ml ampicillin. Single colonies were grown overnight at 37 ° C, and 1 ml of LB medium containing 50 μg / ml ampicillin was inoculated with them in test tubes. Bacteria in test tubes were grown overnight at 30 ° C with shaking at 250 rpm. Then, the grown bacteria, 40 μl each, were inoculated with another set of tubes containing 1 ml LB with ampicillin (50 μg / ml). Samples were then subjected to the steps of inducing expression, harvesting the cell culture, the freeze-thaw treatment cycle, and testing for antiproliferative or antiviral activity, as described above for the initial screening steps.

In each round of screening steps, after confirmation testing, approximately 20 colonies with the highest antiproliferative or antiviral activity were selected to obtain plasmids and their inserts were sequenced automatically. The inserts having a unique DNA sequence were further amplified using a pair of PCR primers, BVBF: 5'-ACCATGAAGGTGACGCTC-3 '(SEQ ID No: 17) and BVR: 5'-AATCTTCTCTCATCCGC-3' (SEQ ID No: 16), which are the upstream and downstream flanking sequences of the multiple cloning sites of the pBVB vector, respectively. Amplified PCR products were used for the next round of shuffling library design.

Based on an increase in either antiproliferative or antiviral activity, five cycles of screening steps were performed.

Example 4. Expression of recombinant Novaferon protein in E. coli and its purification

The Novaferon protein (SEQ ID No: 2) was expressed in E. coli. The reading frame of SEQ ID No: 1 with artificial addition of the ATG initiation codon was cloned in the pBVB thermally inducible vector under the control of the λPRPL promoter (114). Expressing plasmid Novaferon, pBVBNF, transformed DH5α cells. Single colonies were individually selected, they were inoculated with 2 ml of LB medium containing 50 μg / ml ampicillin, and incubated at 30 ° C for 8 hours. Then, 2 ml of cultured bacteria were additionally incubated with 50 ml of medium containing 50 μg / ml ampicillin overnight at 30 ° C with stirring. The next morning, bacteria cultured overnight were seeded at a ratio of 1: 10 ~ 1: 20 in a large volume of LB medium containing 50 μg / ml ampicillin and incubated at 30 ° C with stirring. When the cultures reached the middle of the logarithmic growth phase (A ₅₅₀ = 0.5-0.6), the incubation temperature was quickly raised to 42 ° C and kept for 4 hours in order to induce Novaferon expression. At the end of the 4-hour thermal induction, the bacterial cells were centrifuged and washed 3 times with PBS (phosphate buffered saline) (137 mM NaCl; 2.7 mM KCl; 10 mM Na ₂ HPO ₄ ; 2 mM KH ₂ PO ₄ ), then stored at -80 ° C before cleaning.

Most Novaferon protein molecules were soluble in the E. coli production system described herein, despite being overexpressed into the cytoplasm. Therefore, cells were destroyed by digesting with lysozyme in buffer I for cell lysis (50 mM Tris-Cl (pH 8.0); 1 mM EDTA (ethylenediaminetetraacetic acid) (pH 8.0); 100 mM NaCl) (115). The lysate was further sonicated to destroy the remaining intact cells and splice the DNA molecules. Then the lysate was subjected to centrifugation.

Soluble Novaferon protein molecules in supernatants were sequentially purified by hydrophobic, ion-exchange chromatography and gel filtration. First, supernatants were applied to the column and passed through the column with Phenyl-Sepharose 6 Fast Flow (GE Healthcare, US). Secondly, fractions containing Novaferon protein were applied to a POROS 50 D column (Applied Biosystems, US). Thirdly, the fractions containing Novaferon molecules were purified on a POROS 50 HS column (Applied Biosystems, US) and the finally collected Novaferon molecules were further purified on a HiLoad 26/100 Superdex 75pg (Amersham, US).

The purity of the purified Novaferon protein was checked by analysis by electrophoresis in 15% polyacrylamide gel (PAGE) in the presence of sodium dodecyl sulfate (SDS). Purified recombinant protein Novaferon showed a single zone with a molecular mass (MM) of 19-20 kDa. Mass spectrometric analysis showed that the purity of purified Novaferon was more than 98%, and the molecular weight was 19313 daltons, which was identical to the molecular weight predicted from the amino acid sequence in 19315 daltons.

Example 5. Expression of recombinant HuIFN-α2b in E. coli and its purification

The HuIFN-α2b expressing plasmid, pBV2b, contains the cDNA of the coding region of the mature HuIFN-α2b protein (GeneBank accession number: NM_000605), which is under the control of the thermally inducible λ PRPL promoter. HuIFN-α2b expression was carried out following the protocols described by Joseph S and David WR (116).

Expressing plasmid pBV2bF transformed DH5-α cells. Single colonies were selected individually and 2 ml of LB medium containing 50 μg / ml ampicillin were inoculated with them and incubated at 30 ° C for 8 hours. Then, 2 ml of cultured bacteria were additionally incubated with 50 ml of medium containing 50 μg / ml ampicillin overnight at 30 ° C with stirring. The next morning, the bacterial culture was plated at a ratio of 1: 10 ~ 1: 20 in a large volume of LB medium containing 50 μg / ml ampicillin and incubated at 30 ° C with stirring. When the cultures reached the middle of the logarithmic growth phase (A ₅₅₀ = 0.5-0.6), the incubation temperature was quickly raised to 42 ° C and held for 4 hours in order to induce the expression of HuIFN-α2b. At the end of the 4-hour thermal induction, the bacterial cells were centrifuged and washed with PBS (137 mM NaCl; 2.7 mM KCl; 10 mM Na ₂ HPO ₄ ; 2 mM KH ₂ PO ₄ ), then stored at -80 ° C until purification .

The HuIFN-α2b protein was expressed in insoluble form in the E. coli production system described herein, and therefore, the extraction and washing methods of inclusion bodies were used in accordance with the protocols described in Molecular Cloning (115). Briefly, the collected bacterial cells were resuspended in cell lysis buffer I (50 mM Tris-Cl (pH 8.0); 1 mM EDTA (pH 8.0); 100 mM NaCl) and lyso lyzed and sonicated. Inclusion bodies were washed three times with ice-cold buffer II for cell lysis (buffer I for cell lysis supplemented with 0.5% (v / v) Triton X-100).

The recovered inclusion bodies were destroyed by suspending in 7N. guanidine at room temperature with stirring for 4 hours. After centrifugation at 4 ° C for 15 minutes, the denatured protein was refolded in 0.15 M (pH 9.5) Borex buffer for 48 hours at 4 ° C. In the last refolding step, the pH was adjusted to 7.4 using HCl.

The solution containing the refolded HuIFN-α2b was then purified by hydrophobic and ion exchange chromatography and gel filtration. First, the solution was applied to a column and passed through a column of Phenyl-Sepharose 6 Fast Flow (GE Healthcare, US). Secondly, fractions containing HuIFN-α2b were applied to a POROS 50 D column (Applied Biosystems, US). Thirdly, fractions containing HuIFN-α2b were purified on a POROS 50 HS column (Applied Biosystems, US). The finally assembled HuIFN-α2b molecules were further purified on a HiLoad 26/100 Superdex 75pg (Amersham, US). The purified HuIFN-α2b protein showed a single zone when analyzed by 15% SDS page electrophoresis in the presence of SDS, and its purity was more than 98%, as confirmed by mass spectrometry.

Example 6. Determination of the antiviral activity of Novaferon

Antiviral activity was determined using the WISH-VSV system as described in the classic protocols given by Armstrong JA (113). On the first day, WISH cells (ATCC, CCL 25 catalog number) were seeded in 96-well plates at a density of 14,000 cells / well and incubated at 37 ° C. After 24 hours, 2-fold serial dilutions of Novaferon, HuIFN-α2b, international BO3 standards for human IFN or culture medium comparison were added to each well and incubated at 37 ° C for the next 24 hours. On the third day, the medium was removed and replaced with a medium containing 1000 PFU (plaque forming units) of the virus of vesicular stomatitis (VSV, ATCC, catalog number VR-1421). Cells were again incubated for 24 hours at 37 ° C and then washed with 0.85% NaCl to remove dead cells. The culture plates were then immersed in a fixative dye solution (0.5% crystal violet, 5% formalin (v / v), 50% ethanol (v / v) and 0.85% NaCl) on 1 hour. The fixative dye solution was then removed, the microplates washed extensively with tap water and allowed to dry. Stained cells were dissolved using 0.2 ml of 2-methoxyethanol. Plates were read at 550 nm in Model Opsys MR (Thermo Labsystems, US) for biological analysis of crystalline violet.

All experiments were performed in triplicate, and Novaferon and HuIFN-α2b samples were tested in the same plate. The antiviral activity obtained in this example, Novaferon and HuIFN-α2b were evaluated in parallel and antiviral units (international unit or IU) were determined with reference to international standards BO3, 94/786 (rHuIFN-α consensus) and 95/650 (rHuIFN-α2a), which were acquired at the National Institute of Biological Standards and Control (NIBSC, USA).

The measured antiviral activity of the purified Novaferon protein against VSV on WISH cells was 2.5 × 10 ⁹ IU / mg, while the antiviral activity of HuIFN-α2b was 2.0 × 10 ⁸ IU / mg. These data indicate that the antiviral activity of Novaferon protein is approximately 12.5 times stronger than that of HuIFN-α2b.

Example 7. Antiproliferative activity of Novaferon

The analysis of antiproliferative activity was carried out mainly as described by Evinger and Pestka (101).

A. Cell culture of human tumor cell lines

Human tumor cell lines were acquired at various organizations (Table 1, below), namely ATCC (American Type Culture Collection, PO Box 1549, Manassas, VA 20108, USA), DSMZ (German National Resource Center for Biological Material, Deutsche Sammlung von Mikroorganismen und Zeilkulturen GmbH, Mascheroder Weg Ib, 38124 Braunschweig, Germany), JCRB (Japanese Collection of Research Bioresources-Cell Bank, National Institute of Biomedial Innovation, 7-6-8 Saito-Asagi, Ibaraki-shi, Osaka 567-0085, Japan )

Table 1. Human tumor cell lines Cell lines Tumors Codes Organizations * A-375 Melanoma CRL1619 ATCC IGR-1 Melanoma Acc 236 DSMZ IGR-37 Melanoma Acc 237 DSMZ IPC-298 Melanoma Acc 251 DSMZ HCT-8 Colorectal adenocarcinoma CCL-244 ATCC SW1116 Colorectal adenocarcinoma CCL-233 ATCC LS 180 Colorectal adenocarcinoma CL-187 ATCC DLD-1 Colorectal adenocarcinoma CCL-221 ATCC LS174T Colorectal adenocarcinoma CL-188 ATCC Hepg2 Hepatic Cell Carcinoma HB-8065 ATCC Ner3B Hepatic Cell Carcinoma HB-8064 ATCC Huh-7 Hepatoma 0403 Jcrb PLC / PRF / 5 Hepatoma CRL-8024 ATCC HL60 (S) Lymphocytic forms 0163 Jcrb Daudi Burkitt's Lymphoma CCL-213 ATCC L-428 Hodgkin's lymphoma Acc 197 DSMZ DU 145 Prostate cancer NTV-81 ATCC RS-3 Prostate cancer Acc 465 DSMZ MKN 1 Stomach adenocarcinoma 0252 Jcrb KYSE 30 Esophageal carcinoma 0188 Jcrb A549 Lungs' cancer CCL-185 ATCC Hela Cervical adenocarcinoma CCL-2 ATCC S-33A Cervical cancer HTB-31 ATCC * DSMZ: German National Resource Center for Biological Material (Deutsche Sammlung von Mikroorganismen und Zeilkulturen), Germany ATCC: American Type Culture Collection, USA JCRB: Japanese Collection of Research Bioresources-Cell Bank, Japan

All cells used in the antiproliferative activity test were cultured at 37 ° C. in a humidified atmosphere containing 5% CO ₂ . Cells were grown in accordance with the guidelines for growing each type of cell on basic growth media such as DMEM (Dulbecco's Modified Eagle Medium), MEM (minimal essential medium), F12K and 1640 or 1640 plus F12 (all from Gibco BRL, US) supplemented with 5-20% heat inactivated fetal calf serum (FBS) from Gibco BRL, US. The main media for the growth of each individual cell line are shown in Table 2 below. All cell lines were individually inspected daily on culture plates under an inverted microscope. Cells were collected and used for experiments in their logarithmic growth phase with a vitality exceeding 90%, which was determined by the exclusion of trypan blue dye. Cell counts and determination of viability was carried out in a standard hemocytometer.

Table 2. Cultivation methods and evaluation of human tumor cell lines Cell line Culture media Cells / well Evaluation Methods IGR-1 DMEM 5000 crystal violet bioanalysis IGR-37 DMEM 2000 crystal violet bioanalysis IPC-298 1640 2000 crystal violet bioanalysis HCT-8 1640 500 crystal violet bioanalysis LS 180 Mem 3000 crystal violet bioanalysis DLD-1 1640 1500 crystal violet bioanalysis Hep g2 Mem 1000 crystal violet bioanalysis Ner 3B Mem 800 crystal violet bioanalysis Huh-7 DMEM 4000 crystal violet bioanalysis PLC / PRF / 5 Mem 6000 crystal violet bioanalysis KYSE 30 1640 + F12 1000 crystal violet bioanalysis DU 145 Mem 1000 crystal violet bioanalysis PC-3 1640 2000 crystal violet bioanalysis MKN 1 1640 2000 crystal violet bioanalysis A549 F12k 400 crystal violet bioanalysis

SW1 116 1640 1000 crystal violet bioanalysis LS174T Mem 4000 crystal violet bioanalysis Hela Mem 500 crystal violet bioanalysis S-33A Mem 1000 crystal violet bioanalysis A375 DMEM 200 crystal violet bioanalysis HL 60 (S) 1640 800 direct cell count Daudi 1640 400 direct cell count L-428 1640 800 direct cell count

B. Methodology for the analysis of antiproliferative activity

Cell lines in the logarithmic growth phase were carefully suspended in heated (36 ° C) media to a density of 2 × 10 ³ -6 × 10 ⁴ cells / ml (depending on the cell line, see Table 2). 100 μl of cell suspension were sown in each well of a 96-well plate, followed by incubation for 6-8 hours at 37 ° C. Then, equal volumes (100 μl) of Novaferon or HuIFN-α2b diluted in culture medium were added to the wells in triplicate. The contents of the tablets were carefully mixed for 4-5 seconds and incubated at 37 ° C for 6 days. Samples of Novaferon and HuIFN-α2b were tested in the same tablet in order to guarantee comparability.

Two methods were used to determine the number of cells in the cell-containing well and to calculate the antiproliferative activities of Novaferon and HuIFN-α2b corresponding to these cell numbers.

To determine the number of suspended cells, a direct cell counting method was used. After 6 days of cultivation, suspended cell cultures were diluted with trypan blue (final concentration: 0.02%) and the number of cells was counted directly using a hemocytometer.

To determine the number of adhesive cells, a biological analysis method using crystalline violet was used (93). After 6 days of cultivation, dead cells were removed by pipetting with PBS from the culture wells. Next, the wells were filled with a fixative dye solution for staining living cells for 1 hour. The fixative dye solution contained 0.5% crystalline violet, 5% formalin (v / v), 50% ethanol (v / v), and 0.85% NaCl in distilled water. Then the microplates were washed extensively with tap water and allowed to dry. Stained cells were dissolved using 0.2 ml of 2-methoxyethanol. Optical density was measured at 550 nm (OD ₅₅₀ ) (Model Opsys plate reader, Thermo Labsystems, US) and used as a relative indicator of cell numbers.

The degree of growth inhibition was calculated by the following formula:

the degree of inhibition in% = (1- (EB) / (CB)) X100, where E was the number of cells or OD value ₅₅₀ in the wells treated with Novaferon or HuIFN-α2b on the 6th day; In was the number of cells or the magnitude of OD ₅₅₀ in cell culture on day 0; C was the number of cells or the OD value of ₅₅₀ in untreated wells on day 6.

The degree of inhibition was expressed through the concentration of the compounds. IC ₅₀ Novaferon or HuIFN-α2b was evaluated using a number of samples with different concentrations. The data were fitted to a sigmoid curve (117) with a Hill slope of: Y = Min + (Max-Min) / (1 + 10 ^ (IC ₅₀ -X)), where X is the log of drug concentrations; Y represents the degree of inhibition; Min or Max represents the minimum or maximum degree of inhibition corresponding to a plateau. You can compare the IC ₅₀ for various compounds relative to a specific target, while a lower IC ₅₀ indicates a more potent compound.

The concentrations of Novaferon and HuIFN-α2b and the corresponding degrees of inhibition of cell growth for the Daudi cell line are presented in Figure 4. Based on these data, IC ₅₀ Novaferon and HuIFN-α2b with respect to inhibition of Daudi cell growth were calculated as 0.0174 nmol and 6.9550 nmol. Thus, Novaferon's IC ₅₀ is approximately 1/400 of that of HuIFN-α2b, characterizing approximately 400-fold increase in the antiproliferative efficacy of Novaferon compared to HuIFN-α2b.

The antiproliferative activities of Novaferon were evaluated and compared with those for HuIFN-α2b on 23 tumor cell lines, including 4 cell lines derived from melanoma (A-375, IGR-1, IGR-37, IPC-298), 5 cell lines of colorectal adenocarcinoma ( HCT-8, SW1116, LS 180, DLD-1, LS174T), 4 cell lines of liver cancer (Hep G2, Hep 3B, HuH-7, PLC / PRF / 5), 3 cell lines of lymphoma (HL-60 (S ), Daudi, L-428), 2 cell lines of prostate cancer (DU 145, PC-3), 2 cell lines of cervical cancer (HeLa, C-33A), 1 cell line of gastric adenocarcinoma (MKN 1), 1 cell line lung cancer (A 549) and one cl the exact line of esophageal cancer (KYSE 30). Novaferon exhibits stronger antiproliferative activity compared to HuIFN-α2b for all tested cancer cell lines. The degree of increase in efficiency varied for different cancer cell lines and varied with a ratio of 16 to 1134 (Table 3, below).

Table 3. IC ₅₀ values for Novaferon and HuIFN-α2b and the increase in the inhibition of tumor cells by Novaferon compared to HuIFN-α2b Cell lines IC ₅₀ (nmol) Multiplicity HuIFN-α2b Novaferon (Novaferon / HuIFN-α2b) PLC / PRF / 5 0,0407 0.0025 16 A549 4.27 0.2202 19 DU 145 0.1319 0.0036 36 Hepg2 0.1718 0.004 43 Huh-7 0.1474 0.0026 58 Hep3b 4.3934 0,0758 58 IPC-298 0,0516 0,0007 70 LS174T 0.0165 0,0002 74 IGR-37 0.6017 0.0055 109 PC-3 1.8777 0.0146 128 Hela 0.2364 0.0017 141 C-33a 2,5242 0.0176 143 MKN 1 0.233 0.0011 207 HCT-8 2,9479 0.0139 212 SW1116 0.2278 0.001 222 DLD-1 0.3977 0.0014 282 HL-60 (S) 0.5855 0.0019 306 LS 180 0.7579 0.0022 350 Daudi 6,955 0.0174 400 KYSE 30 18,0134 0.0264 683 A-375 1.4134 0.0019 733 IGR-1 6.6718 0.0076 876 L-428 17,2789 0.0152 1134

Example 8. Experiments on in vivo tumor models

A. Cell culture and xenograft models of human tumors in vivo

Colon cancer cell line (LS 180), melanoma cell line (A-375), and liver cancer cell line (Hep G2) were obtained from the American Type Culture Collection (ATCC, Rockville, MD). A prostate cancer cell line (PC-3) was obtained from the German National Resource Center for Biological Material (DSMZ, Deutsche Sammlung von Mikroorganismen und Zeilkulturen, Germany). The lymphocytic cell line (HL 60 (s)) was purchased from the Japanese Collection of Research Bioresources Cell Bank (JCRB, Japan). All cells were cultured in accordance with the instructions for them (see Table 1). Briefly, LS 180 and Hep G2 were cultured in MEM medium. A-375 was cultured in DMEM. Both media were supplemented with 10% fetal calf serum (FBS), 2 mM glutamine, 100 units / ml penicillin, 100 mg / ml streptomycin, 0.1 mM non-essential amino acids and 1.0 mM sodium pyruvate. PC-3 and HL 60 (8) cells were cultured in RPMI 1640 (Medium 1640 from Roswell Park Memorial Institute) supplemented with 10% FBS, 100 units / ml penicillin and 100 mg / ml streptomycin. All cells were maintained in an atmosphere of 5% CO ₂ at 37 ° C.

Xenograft models of human tumors were created using methods described by Beverly et al. (118). Cancer cells in the logarithmic growth phase were collected from tissue culture plates, washed and resuspended in phosphate buffered saline (PBS, pH = 7.5; 20 mM). 6-week old Balb / c nude mice generated subcutaneous tumor xenografts by injection of 6 × 10 ⁶ cells / 0.3 ml (PC-3, HepG2), 4 × 10 ⁶ cells / 0.3 ml (LS 180), 2 × 10 ⁷ cells / 0.3 ml (HL 60 (s)) or 8 × 10 ⁶ cells / 0.3 ml (A-375) subcutaneously on both sides of the lateral region. For each in vivo tumor model, on the 6th day after inoculation of tumor cells of tumor-bearing mice (tumor volume is approximately 100 mm ³ ), they were randomly divided into 7 or 8 groups with the same number of animals in each group and treatment was started.

Novaferon and HuIFN-α2b were prepared using a PBS solution. Daily subcutaneous injections of only PBS, various doses of Novaferon or HuIFN-α2b were continued for a total of 30 days (PC-3, HepG2, A-375), 28 days (LS 180) or 21 days (HL 60 (s)), starting from the day of the formation of groups of mice. For treatment using 5-FU, once every two days, a total of 5 times, 30 mg / kg of 5-FU was administered iv. The groups and dosage are summarized below.

Group 1 (control): PBS daily.

Group 2 (low dose Novaferon): 1.25 mcg / kg daily.

Group 3 (average dose of Novaferon): 12.5 mcg / kg daily.

Group 4 (high dose of Novaferon): 125 mcg / kg daily.

Group 5 (low dose of HuIFN-α2b): 1.25 mcg / kg daily.

Group 6 (average dose of HuIFN-α2b): 12.5 mcg / kg daily.

Group 7 (high dose of HuIFN-α2b): 125 mcg / kg daily.

Group 8 (5-FU): 30 mg / kg, iv once every 2 days, 5 times.

After the start of treatment, tumors were measured using calipers once a week. Tumor volumes were calculated using the following formula: volume = 0.5 × (width) ² × (length). Mice were euthanized on the day the treatment was completed (30th day after the start of treatment). Solid tumors were removed, photographed and measured.

The degree of growth inhibition was calculated using the following formula: degree of inhibition = [1-T / C] × 100%, where T represents the average tumor weight in Novaferon, HuIFN-α2b or 5-FU-treated groups; C represents the average tumor weight in the control group after treatment.

B. Model of a human prostate cancer xenograft

Xenografts of the PC-3 prostate cancer were treated with subcutaneous (subcutaneous) injection of 1.25, 12.5 or 125 μg / kg of Novaferon for 30 days. Novaferon showed strong, dose-dependent inhibition of PC-3 tumor growth (P <0.05). As shown in FIG. 5 and Table 4 below, the growth of PC-3 tumor in Novaferon-treated groups was more strongly inhibited compared with the control group treated with PBS. For example, the average weight of the tumor mass of PC-3 xenograft in the Novaferon-treated group (125 μg / kg), 0.091 ± 0.081 g, was very significantly reduced compared to control animals, 1.948 ± 0.567 g (P <0.001) (Table 4) . In other words, as a result of 30-day treatment with 125 μg / kg, 95.3% inhibition of tumor growth of PC-3 was achieved (Table 4).

Table 4. Tumor weight and the degree of inhibition of the growth of xenografts of the PC-3 human prostate cancer treated with Novaferon and HuIFN-α2b (n = 10) Group Dose (mcg / kg) Tumor Weight (g) (mean ± SD) The degree of inhibition (%) The control - 1.948 ± 0.567 Novaferon, low dosage 1.25 1.266 ± 0.457 * 35.0 Novaferon, average dosage 12.5 0.759 ± 0.574 *** 61.0 Novaferon, high dosage 125 0,091 ± 0,081 *** @@@ 95.3 HuIFN-α2b, low dosage 1.25 1.284 ± 0.862 34.1 HuIFN-α2b, average dosage 12.5 0.790 ± 0.391 *** 59,4 HuIFN-α2b, high dosage 125 0.476 ± 0.271 *** 75.6 Note: * p <0.05; *** p <0.001: compared with the control group; @@@ p <0.001: compared with the HuIFN-α2b group, high dose. SD: standard deviation.

Nude athymic Balb / c mice were treated daily by sc injection of Novaferon (1.25 μg / kg, 12.5 μg / kg or 125 μg / kg) for 30 days after subcutaneous administration to mice of 6 × 10 ⁶ live PC-3 cells . The results were expressed as the average tumor volume (mm ³ ). Figure 5 shows that all three doses of Novaferon showed a dose-dependent inhibition of tumor growth of PC-3 compared with the control group PBS (P <0.05). 125 μg / kg Novaferon induced a very strong or almost complete inhibition of PC-3 tumor growth compared to that for HuIFN-α2b at the same dose (95.3% versus 75.6%, P <0.01) (Table 4) .

It is interesting to note that a longer treatment with Novaferon or HuIFN-α2b led to more noticeable differences in the inhibition of tumor growth in the groups treated with a large dose (125 μg / kg). The mean PC-3 tumor mass volume in the Novaferon-treated group was 107.9 ± 68.7 mm ³ compared with 620.7 ± 296.6 mm ³ in the HuIFN-α2b-treated group on day 28 (P <0.001 ) and 122.1 ± 100.7 mm ³ compared with 691.9 ± 428.3 mm ³ on the 30th day (P <0.001). This also applies to the case when the average tumor weight was considered after observation (0.091 ± 0.081 g for the high dose Novaferon group compared to 0.476 ± 0.271 g for the high dose group HuIFN-α2b, P <0.001). This confirms that a longer treatment with Novaferon at this dose may demonstrate better or complete inhibition of tumor growth of PC-3 in this xenograft model.

C. Human liver cancer xenograft model

The in vivo antitumor activity of Novaferon was also evaluated in a liver cancer Hep G2 xenograft model. Novaferon showed an effective, dose-dependent inhibition of tumor growth of Mer G2 compared with the control group (P <0.001). The average tumor volumes in Novaferon-treated groups (daily subcutaneous injection of 1.25, 12.5 or 125 μg / kg for 30 days) were 783.2 ± 270.0, 459.3 ± 414.3 and 104.6 ± 56.5 mm ³ , respectively, compared with 2125.8 ± 743.1 mm ³ in the PBS control group. As a result of a 30-day treatment, 125 μg / kg of Novaferon achieved the highest inhibition of Hep G2 (96.6%), which was more significant than that for 125 μg / kg of HuIFN-α2b (89.2%, P <0.01 ) Longer treatment with Novaferon at this dose showed a tendency to even better or complete inhibition. The average tumor weight at the end of the observation period was 0.074 ± 0.083 g for the Novaferon-treated group (125 μg / kg), which is significantly lower than that for the HuIFN-α2b-treated group (125 μg / kg) (0.235 ± 0.199 g, P <0.001) (Table 5, below).

Nude athymic Balb / c mice were treated daily by sc injection of Novaferon (1.25 μg / kg, 12.5 μg / kg and 125 μg / kg) for 30 days after subcutaneous administration to mice of 6 × 10 ⁶ live Hep G2 cells. The results were expressed as the average tumor volume (mm ³ ). Figure 6 shows that all three doses of Novaferon showed a dose-dependent inhibition of tumor growth Hep G2 compared with the control group PBS (P <0.001). 125 μg / kg Novaferon induced a very strong or almost complete inhibition of Hep G2 tumor growth compared to that for HuIFN-α2b at the same dose (96.6% versus 89.2%, P <0.05) (Table 5) .

Table 5. Tumor weight and the degree of inhibition of the growth of xenografts Nep G2 human liver cancer treated with Novaferon and HuIFN-α2b (n = 10) Group Dose (mcg / kg) Tumor Weight (g) (mean ± SD) The degree of inhibition (%) The control - 2.179 ± 0.578 - Novaferon, low dosage 1.25 0.797 ± 0.397 *** 63,4

Novaferon, average dosage 12.5 0.321 ± 0.300 *** 85.3 Novaferon, high dosage 125 0.074 ± 0.083 *** @ 96.6 HuIFN-α2b, low dosage 1.25 1,070 ± 0,587 ** 50.9 HuIFN-α2b, average dosage 12.5 0.531 ± 0.287 *** 75.6 HuIFN-α2b, high dosage 125 0.235 ± 0.199 *** 89.2 Note: ** p <0.01; *** p <0.001: compared with the control group; @ p <0.01: compared with the HuIFN-α2b group, a high dose.

D. Model of human melanoma xenograft

The in vivo antitumor activity of Novaferon was additionally evaluated using the A-375 xenograft model of human malignant melanoma. Cell line A-375 (ATCC number: CRL-1619) was removed from a human malignant solid tumor. Novaferon showed an effective, dose-dependent inhibition of tumor growth of A-375 compared with the control group (P <0.001). The degree of inhibition in Novaferon-treated groups (daily SC injection of 1.25, 12.5 or 125 μg / kg for 28 days) was 40.1%, 75.0% and 82.8%, respectively, compared with the PBS control group (P <0.001) (Table 6, below).

As a result of a 30-day treatment, 125 μg / kg of Novaferon achieved the highest inhibition of A-375 (82.8%), which was more significant than that for 125 μg / kg of HuIFN-α2b (69.9%; P <0.001) .

Interestingly, Novaferon showed a more effective inhibition of the growth of A-375 melanoma cells compared with the chemotherapeutic agent 5-FU (Table 6). On the 30th day, for example, the average weight of tumors in the groups treated with 12.5 μg / kg or 125 μg / kg of Novaferon was 0.763 ± 0.187 (P <0.01) and 0.527 ± 0.149 (P <0.001) g, while the average tumor weight in the group treated with 5-FU, 30 mg / kg, was 1.004 ± 0.105 g (Table 6). This suggests that Novaferon may be more effective in treating human A-375 melanoma than 5-FU.

Nude nude Balb / c mice were treated daily by sc injection of Novaferon (1.25 μg / kg, 12.5 μg / kg and 125 μg / kg) for 28 days after subcutaneous administration of 8 × 10 ⁶ A-375 cells to mice. The results were expressed as the average tumor volume (mm ³ ). Figure 7 shows that all three doses of Novaferon showed dose-dependent inhibition of tumor growth of A-375 compared with the control group PBS (P <0.001). 125 μg / kg Novaferon induced a stronger inhibition of tumor growth of A-375 compared to that for HuIFN-α2b at the same dose (82.8% versus 69.9%, P <0.001) (Fig. 7). Both 12.5 μg / kg and 125 μg / kg of Novaferon showed better suppression (75.0% and 82.8%, respectively) of tumor growth than 5-FU (67.2%, P <0.01 and P <0.001) (Fig. 7).

Table 6. Tumor weight and the degree of inhibition of xenograft growth of human melanoma A-375 cells treated with Novaferon and HuIFN-α2b (n = 10) Group Dose (mcg / kg) Tumor Weight (g) (X ± SD) The degree of inhibition (%) The control - 3,057 ± 0,384 Novaferon, low dosage 1.25 1.830 ± 0.289 *** 40.1 Novaferon, average dosage 12.5 0.763 ± 0.187 *** && $$$ 75.0 Novaferon, high dosage 125 0.527 ± 0.149 *** &&& @@@ 82.8 HuIFN-α2b, low dosage 1.25 1.890 ± 0.148 *** 38,2 HuIFN-α2b, average dosage 12.5 1.681 ± 0.132 *** 45.0 HuIFN-α2b, high dosage 125 0.920 ± 0.139 *** 69.9 5-fu 30000 1.004 ± 0.105 *** 67.2 Note: *** p <0.001: compared with the control group; $$$ p <0.001: compared to HuIFN-α2b, average dose (12.5); @@@ p <0.001: compared with the HuIFN-α2b group, high dose (125); &&: p <0.01, &&&: p <0.001: compared to the 5-FU group.

E. Model of human colon cancer xenograft

The in vivo antitumor activity of Novaferon was additionally evaluated in a colon cancer xenograft LS 180 model. The LS 180 cell line (ATCC number: CL-187) was removed from human colon adenocarcinoma. Novaferon showed an effective, dose-dependent inhibition of LS 180 tumor growth compared with the control group (P <0.001). The degree of inhibition in Novaferon-treated groups (daily SC injection of 1.25, 12.5 or 125 mcg / kg for 28 days) was 75.0%, 80.5% and 92.5%, respectively, compared with a PBS control group (P <0.001) (Table 7, below). As a result of 28-day treatment with 125 μg / kg, Novaferon achieved the highest tumor growth inhibition LS 180 (92.5%), which was more significant compared to 125 μg / kg HuIFN-α2b (82.3%, P <0.001 )

After 28 days of treatment with Novaferon (12.5 μg / kg), LS 180 cancer xenograft growth was inhibited similarly to 5-FU (30 mg / kg) in terms of average tumor weight (0.815 ± 0.221 g compared to 0.758 ± 0.227 g). 125 μg / kg of Novaferon inhibited tumor growth of LS 180 significantly better than 30 mg / kg of 5-FU (92.5% compared with 81.8%, P <0.001) (Table 7 and Fig. 8). These observations were extremely interesting, given the generally accepted clinical use of 5-FU in standard chemotherapy for patients with colon cancer. The best Novaferon suppression of LS 180 tumor growth in an animal model shows that Novaferon has the potential to be a very effective agent against colon cancer in a clinical setting.

Nude athymic Balb / c mice were treated with a daily injection of Novaferon (1.25 μg / kg, 12.5 μg / kg and 125 μg / kg) for 28 days after subcutaneous administration to mice of 4 × 10 ⁶ LS 180 cells. The results were expressed as the average tumor volume (mm ³ ). On Fig shows that all three doses of Novaferon showed a dose-dependent inhibition of tumor growth LS180 compared with the control group PBS (P <0.001). 125 μg / kg Novaferon induced a stronger inhibition of tumor growth LS 180 than that for HuIFN-α2b at the same dose (92.5% versus 82.3%, P <0.001) (table 7). Using both 1.25 μg / kg and 12.5 μg / kg, Novaferon achieved a similar inhibition (75.0% and 80.5%, respectively) of tumor growth compared to 5-FU (81.8%) ( Table 7, Fig. 8). However, 125 μg / kg of Novaferon showed a significantly stronger inhibition of tumor growth LS 180 than that under the action of 5-FU (92.5% versus 81.8%, P <0.001).

Table 7. Tumor weight and degree of inhibition of xenograft growth LS 180 human colon cancer cells treated with Novaferon and HuIFN-α2b (n = 10) Group Dose (mcg / kg) Tumor Weight (g) (X ± SD) The degree of inhibition (%) The control - 4,170 ± 3,409 - Novaferon, low dosage 1.25 1.043 ± 0.433 *** 75.0 Novaferon, average dosage 12.5 0.815 ± 0.221 *** 80.5 Novaferon, high dosage 125 0.314 ± 0.086 *** &&& @@@ 92.5 HuIFN-α2b, low dosage 1.25 1.225 ± 0.565 *** 70.6 HuIFN-α2b, average dosage 12.5 1,076 ± 0,442 *** 74,2

HuIFN-cr2b high dosage 125 0.740 ± 0.310 *** 82.3 5-fu 30000 0.758 ± 0.227 *** 81.8 Note: *** p <0.001: compared with the control group; @@@ p <0.001: compared to HuIFN-α2b, high dosage; &&& p <0.001: compared to the 5-FU group.

F. Human Leukemia Xenograft Model

The in vivo antitumor activity of Novaferon was also evaluated in the HL 60 (s) lymphocytic leukemia xenograft model. Novaferon showed effective, dose-dependent inhibition of HL 60 (s) tumor growth compared with the control group (P <0.001). The degrees of inhibition in Novaferon-treated groups (daily SC injection of 1.25, 12.5 or 125 μg / kg for 28 days) were 43.8%, 55.2% and 80.4%, respectively, compared with the PBS control group (P <0.001, Table 8, below). After 21 days of treatment with Novaferon (125 μg / kg), the highest inhibition of HL 60 (s) tumor growth (80.4%) was achieved, which was significantly better than that with 125 μg / kg HuIFN-α2b (69.8% , P <0.05).

Balb / c mice were treated daily by sc injection of Novaferon (1.25 μg / kg, 12.5 μg / kg and 125 μg / kg) for 21 days after subcutaneous administration to mice of 2 × 10 ⁷ live HL 60 (s) cells . The results were expressed as the average tumor volume (mm ³ ). Figure 9 shows that all three doses of Novaferon showed a dose-dependent inhibition of tumor growth HL 60 (s) compared with the control group PBS (P <0.001). 125 μg / kg of Novaferon induced a stronger inhibition of HL 60 (s) tumor growth than that induced by HuIFN-α2b at the same dose (80.4% versus 69.8%, P <0.05), and a similar inhibition, comparable to that for 5-FU (Fig. 9, Table 8).

Table 8. Tumor weight and degree of growth inhibition of HL 60 (S) xenografts of human leukemia cells treated with Novaferon and HuIFN-α2b (n = 10) Group Dose (mcg / kg) Tumor Weight (g) (X ± SD) The degree of inhibition (%) The control - 3,723 ± 0,750 - Novaferon, low dosage 1.25 2.091 ± 0.653 *** 43.8 Novaferon, average dosage 12.5 1.668 ± 0.665 *** 55,2 Novaferon, high dosage 125 0.729 ± 0.332 *** @ 80,4 HuIFN-α2b, low dosage 1.25 2,401 ± 0,698 *** 35.5 HuIFN-α2b, average dosage 12.5 1.870 ± 0.660 *** 49.8

HuIFN-or2b, high dosage 125 1.124 ± 0.397 *** 69.8 5-fu 30000 0.893 ± 0.289 *** 76.0 Note: *** p <0.001: compared with the control group; @ p <0.05: compared to the HuIFN-α2b group, high dosage (125).

G. General condition of mice during treatment with Novaferon

Mice with various xenografts of human cancer were carefully monitored during the treatment period with Novaferon, HuIFN-α2b or 5-FU. Unlike 5-FU-treated groups, mice in all Novaferon or HuIFN-α2b-treated groups usually fed and behaved normally and did not show weight loss. 5-FU-treated mice showed typical changes in feed intake and behavior and weight reduction. These observations indicate that although Novaferon exhibits similar or better anticancer efficacy than 5-FU in animal xenograft models, it may be more specific for cancer cell inhibition and have less effect on normal cells and / or physiological functions . This can lead to better tolerance and stronger therapeutic effects in human applications.

As will be apparent to those skilled in the art in light of the above description, when practicing the present invention, many changes and modifications are possible without deviating from its essence or scope. Therefore, the scope of the invention should be construed in accordance with the content defined by the attached claims.

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Claims (49)

1. A protein exhibiting interferon-like antiviral and antiproliferative biological activities, where the specified protein is characterized by an amino acid sequence of at least 89% identical to SEQ ID No: 2. 2. The protein of claim 1, wherein said sequence is at least 90% identical to SEQ ID No: 2. 3. The protein of claim 1, wherein said sequence is at least 95% identical to SEQ ID No: 2. 4. The protein according to any one of claims 1 to 3, where the specified protein has increased antiviral and antiproliferative activity compared to HuIFN-α2b. 5. The protein of claim 4, wherein said protein has antiviral activity at least 2 times greater than HuIFN-α2b. 6. The protein of claim 5, wherein said protein has an antiviral activity of at least 5 times that of HuIFN-α2b. 7. The protein of claim 6, wherein said protein has antiviral activity at least 10 times greater than HuIFN-α2b. 8. The protein of claim 4, wherein said protein has antiproliferative activity of at least 10 times that of HuIFN-α2b. 9. The protein of claim 8, where the specified protein has antiproliferative activity at least 50 times greater than HuIFN-α2b. 10. The protein of claim 9, wherein said protein has antiproliferative activity at least 100 times greater than HuIFN-α2b. 11. The protein of claim 10, wherein said protein has antiproliferative activity of at least 200 times that of HuIFN-α2b. 12. The protein of claim 1, wherein said protein is recombinant. 13. The protein of claim 1, wherein said protein has a molecular weight of 19315 daltons. 14. The protein of claim 1, possibly containing a methionine residue at the N-terminus. 15. The protein according to claim 1, exhibiting interferon-like antiviral and antiproliferative biological activities, where the specified protein has an amino acid sequence of at least 95% identical to SEQ ID No: 2, and where the specified protein has increased antiviral and antiproliferative activities compared to HuIFN -α2b. 16. The protein according to claim 1, exhibiting interferon-like antiviral and antiproliferative biological activities, where the specified protein is characterized by an amino acid sequence of at least 95% identical to SEQ ID No: 2. 17. The protein according to claim 1, containing a sequence that differs from SEQ ID No: 2 in amino acids in an amount of from 0 to 8, where the specified protein exhibits interferon-like antiviral and antiproliferative biological activities. 18. A protein containing a sequence that differs from SEQ ID No: 2 in amino acids in an amount of from 0 to 19, where the protein exhibits interferon-like antiviral and antiproliferative biological activities. 19. The protein of claim 18, wherein said protein has increased antiviral and antiproliferative activity compared to HuIFN-a2b. 20. The protein of claim 19, wherein said protein has an antiviral activity of at least 2 times that of HuIFN-α2b. 21. The protein of claim 18, wherein said protein has antiproliferative activity of at least 10 times that of HuIFN-α2b. 22. The protein of claim 21, wherein said protein has antiproliferative activity at least 100 times greater than HuIFN-α2b. 23. The protein of claim 18, wherein said protein is recombinant. 24. A protein having the amino acid sequence of SEQ ID No: 2 or a fragment thereof containing at least 146 amino acids exhibiting interferon-like antiviral and antiproliferative biological activities. 25. The protein construct containing the protein according to any one of claims 1 to 24, functionally connected to a sugar, peptide or polymer moiety or a labeled molecule, wherein said construct exhibits said interferon-like antiviral and antiproliferative biological activities. 26. The protein construct of claim 25, wherein said protein is glycosylated. 27. The protein construct of claim 25, wherein said moiety is a polypeptide. 28. The protein construct of claim 25, wherein said moiety is not a polypeptide. 29. The protein construct of claim 28, wherein said moiety is a polymer molecule. 30. The protein construct according to clause 29, where the specified polymer molecule is a linear or branched polyethylene glycol. 31. The protein construct of claim 25, wherein said moiety is a labeled molecule. 32. Polynucleotide encoding a protein according to any one of claims 1 to 24. 33. A polynucleotide encoding a protein having interferon-like antiviral and antiproliferative biological activities, wherein said polynucleotide is characterized by a nucleotide sequence of at least 95% identical to SEQ ID No: 1. 34. The polynucleotide of claim 33, wherein said sequence is at least 97% identical to SEQ ID No: 1. 35. The polynucleotide of claim 34, wherein said sequence is at least 98% identical to SEQ ID No: 1. 36. The polynucleotide according to any one of claims 33-35, wherein said protein is non-existent in nature. 37. The polynucleotide according to clause 36, where the specified protein has increased antiviral and antiproliferative activities compared to human interferon-alpha-2b (HuIFN-α2b). 38. The polynucleotide of claim 37, wherein said protein has antiviral activity of at least 2 times that of HuIFN-α2b. 39. The polynucleotide according to clause 37, where the specified protein has antiproliferative activity at least 10 times greater than HuIFN-α2b. 40. The polynucleotide according to claim 33, encoding a protein having interferon-like antiviral and antiproliferative biological activities, wherein said polynucleotide is characterized by a nucleotide sequence of at least 97% identical to SEQ ID No: 1. 41. An expression vector containing a polynucleotide according to any one of claims 33-40. 42. A host cell for producing a protein exhibiting interferon-like antiviral and antiproliferative biological activities, comprising the recombinant vector according to paragraph 41. 43. A composition suitable as an antiviral agent, antiproliferative agent, anticancer agent or immunomodulating agent, containing the protein according to any one of claims 1 to 24 and a pharmaceutically acceptable carrier, diluent or excipient. 44. The composition according to item 43, containing the protein according to item 15. 45. The use of the protein according to any one of claims 1 to 24 as an antiviral agent, antiproliferative agent, anticancer agent or immunomodulating agent. 46. A method of treating a condition susceptible to interferon therapy, cancer or a viral disease, comprising administering to a subject in need of therapy a therapeutically effective amount of a protein according to any one of claims 1-24. 47. The method according to item 46, where the specified subject is a person. 48. The method according to item 46, where the specified protein is administered together with a pharmaceutically acceptable carrier, diluent or excipient. 49. The method of claim 46, wherein said protein is therapeutically effective against a wide range of different types of cancer, wherein said cancer is selected from the group consisting of melanoma, colorectal adenocarcinoma, renal cell cancer, hepatoma, lymphoma, prostate cancer, adenocarcinoma stomach, cancer of the esophagus, lung cancer, cervical adenocarcinoma and cervical cancer.

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